Immunogenic Mycoplasma hyopneumoniae polypeptides

ABSTRACT

Mycoplasma hyopneumoniae  polypeptides and nucleic acids, as well as nucleic acid expression vectors and host cells containing nucleic acid vectors are provided. In addition, compositions containing  M. hyopneumoniae  polypeptides and nucleic acids are provided for use in methods of treating swine to prevent enzootic pneumonia. Furthermore, the invention provides diagnostic tests for the detecting of  M. hyopneumoniae  infection in swine herds.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) of U.S.application No. 60/392,632, filed Jun. 28, 2002.

BACKGROUND

1. Technical Field

The invention relates to methods and materials involved in protecting ananimal against enzootic pneumonia.

2. Background Information

Enzootic pneumonia in swine, also called mycoplasmal pneumonia, iscaused by Mycoplasma hyopneumoniae. The disease is chronic andnon-fatal, affecting pigs of all ages. Although infected pigs show onlymild symptoms of coughs and fever, the disease has significant economicimpact due to reduced feed efficiency and reduced weight gain. Enzooticpneumonia is transmitted by airborne organisms expelled from the lungsof infected pigs. The primary infection by M. hyopneumoniae may befollowed by a secondary infection of other Mycoplasma species, e.g.,Mycoplasma hyorhinis and Mycoplasma flocculare, as well as otherbacterial pathogens.

M. hyopneumoniae infects the respiratory tracts of pigs, colonizing thetracheae, bronchi, and bronchioles. The pathogen produces a ciliostaticfactor that causes the cilia lining the respiratory passages to stopbeating. Eventually, the cilia degenerate, leaving pigs prone toinfection by secondary pathogens. Characteristic lesions of purple togray areas of consolidation are observed in infected pigs. Surveys ofslaughtered pigs revealed lesions in 30% to 80%. Results from 37 herdsin 13 states indicated that 99% of the herds had pigs with pneumonialesions typical of enzootic pneumonia. Therefore, there is a need foreffective preventative and treatment measures.

Mycoplasmas vary their surface structure by a complex series of geneticevents to present a structural mosaic to the host immune system. Phaseswitching of surface molecules occurs through a variety of mechanismssuch as changes in the number of repetitive units during DNAreplication, genomic inversions, transposition events, and/or geneconversion. See, for example, Zhang and Wise, 1997, Mol. Microbiol.,25:859-69; Theiss and Wise, 1997, J. Bacteriol., 179:4013-22; Sachse etal., 2000, Infect. Immun., 68:680-7; Dybvig and Uy, 1994, Mol.Microbiol., 12:547-60; and Lysnyansky et al., 1996, J. Bacteriol.,178:5395-5401. All of the identified phase variable and phase switchinggenes in mycoplasmas that code for surface proteins are lipoproteins.

SUMMARY

The invention provides materials and methods for protecting an animalfrom enzootic pneumonia. The invention is based on the discovery ofMycoplasma hyopneumoniae nucleic acids that encode cell surfacepolypeptides that can be used for inducing a protective immune responsein an animal susceptible to pneumonia. More specifically, the inventionprovides purified immunogenic polypeptides of these polypeptides forused to as antigens for illiciting an immune response in an animal, e.g.a pig. In addition, the invention also provides isolated nucleic acidsencoding these immunogenic polypeptides for use in generating an immuneresponse in an animal. Purified polypeptides and isolated nucleic acidsof the invention can be combined with pharmaceutically acceptablecarriers for introducing into an animal. The invention also providesmaterials and methods for determining whether an animal has an antibodyreactive to the polypeptides of the invention.

In one aspect, the invention provides a purified immunogenicpolypeptide, the amino acid sequence of which comprises at least eightconsecutive residues of a sequence selected from the group consisting ofSEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, and 20. Specifically, theinvention provides an immunogenic polypeptide of the invention, theamino acid sequence of which comprises at least eight consecutiveresidues of SEQ ID NO: 2; an immunogenic polypeptide of the invention,the amino acid sequence of which comprises at least eight consecutiveresidues of SEQ ID NO:4; an immunogenic polypeptide of the invention,the amino acid sequence of which comprises at least eight consecutiveresidues of SEQ ID NO:6; an immunogenic polypeptide of the invention,the amino acid sequence of which comprises at least eight consecutiveresidues of SEQ ID NO:8; an immunogenic polypeptide of the invention,the amino acid sequence of which comprises at least eight consecutiveresidues of SEQ ID NO:10; an immunogenic polypeptide of the invention,the amino acid sequence of which comprises at least eight consecutiveresidues of SEQ ID NO:12; an immunogenic polypeptide of the invention,the amino acid sequence of which comprises at least eight consecutiveresidues of SEQ ID NO:14; an immunogenic polypeptide of the invention,the amino acid sequence of which comprises at least eight consecutiveresidues of SEQ ID NO:16; an immunogenic polypeptide of the invention,the amino acid sequence of which comprises at least eight consecutiveresidues of SEQ ID NO:18; an immunogenic polypeptide of the invention,the amino acid sequence of which comprises at least eight consecutiveresidues of SEQ ID NO: 20.

In another aspect, the invention provides mutants of the above-describedimmunogenic polypeptides, wherein such mutant polypeptides retainimmunogenicity.

Generally, immunogenic polypeptides and immunogenic mutant polypeptidesof the invention include at least 8 consecutive residues (e.g., at least10, 12, 15, 20, or 25) of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, or20.

In another aspect, the invention provides a composition that includesone or more of the above-described immunogenic polypeptides orimmunogenic mutant polypeptides.

In one aspect, the invention provides a method of eliciting an immuneresponse in an animal. Such a method includes introducing a compositioncomprising the above-described immunogenic polypeptides or immunogenicmutant polypeptides into the animal. Such a composition can beadministered orally, intranasally, intraperitoneally, intramuscularly,subcutaneously, or intravenously. A representative animal into which thecompositions of the invention can be introduced is a swine.

In another aspect, the invention provides an isolated nucleic acidcomprising a nucleotide sequence that encodes an immunogenicpolypeptide, the amino acid sequence of which comprises at least eightconsecutive residues of a sequence such as SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 16, 18, or 20. The invention also features mutants of nucleicacids that encode an immunogenic polypeptide. Representative nucleicacids encoding such immunogenic polypeptides have a nucleotide sequenceas shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19,respectively.

Specifically, the invention provides a nucleic acid having a nucleotidesequence encoding an immunogenic polypeptide, the amino acid sequence ofwhich comprises at least eight consecutive residues of SEQ ID NO:2. Arepresentative nucleic acid encoding such a polypeptide has thenucleotide sequence of SEQ ID NO:1.

Specifically, the invention provides a nucleic acid having a nucleotidesequence encoding an immunogenic polypeptide, the amino acid sequence ofwhich comprises at least eight consecutive residues of SEQ ID NO:4. Arepresentative nucleic acid encoding such a polypeptide has thenucleotide sequence of SEQ ID NO:3.

Specifically, the invention provides a nucleic acid having a nucleotidesequence encoding an immunogenic polypeptide, the amino acid sequence ofwhich comprises at least eight consecutive residues of SEQ ID NO:6. Arepresentative nucleic acid encoding such a polypeptide has thenucleotide sequence of SEQ ID NO:5.

Specifically, the invention provides a nucleic acid having a nucleotidesequence encoding an immunogenic polypeptide, the amino acid sequence ofwhich comprises at least eight consecutive residues of SEQ ID NO:8. Arepresentative nucleic acid encoding such a polypeptide has thenucleotide sequence of SEQ ID NO:7.

Specifically, the invention provides a nucleic acid having a nucleotidesequence encoding an immunogenic polypeptide, the amino acid sequence ofwhich comprises at least eight consecutive residues of SEQ ID NO:10. Arepresentative nucleic acid encoding such a polypeptide has thenucleotide sequence of SEQ ID NO:9.

Specifically, the invention provides a nucleic acid having a nucleotidesequence encoding an immunogenic polypeptide, the amino acid sequence ofwhich comprises at least eight consecutive residues of SEQ ID NO:12. Arepresentative nucleic acid encoding such a polypeptide has thenucleotide sequence of SEQ ID NO:11.

Specifically, the invention provides a nucleic acid having a nucleotidesequence encoding an immunogenic polypeptide, the amino acid sequence ofwhich comprises at least eight consecutive residues of SEQ ID NO:14. Arepresentative nucleic acid encoding such a polypeptide has thenucleotide sequence of SEQ ID NO:13.

Specifically, the invention provides a nucleic acid having a nucleotidesequence encoding an immunogenic polypeptide, the amino acid sequence ofwhich comprises at least eight consecutive residues of SEQ ID NO:16. Arepresentative nucleic acid encoding such a polypeptide has thenucleotide sequence of SEQ ID NO:15.

Specifically, the invention provides a nucleic acid having a nucleotidesequence encoding an immunogenic polypeptide, the amino acid sequence ofwhich comprises at least eight consecutive residues of SEQ ID NO:18. Arepresentative nucleic acid encoding such a polypeptide has thenucleotide sequence of SEQ ID NO:17.

Specifically, the invention provides a nucleic acid having a nucleotidesequence encoding an immunogenic polypeptide, the amino acid sequence ofwhich comprises at least eight consecutive residues of SEQ ID NO:20. Arepresentative nucleic acid encoding such a polypeptide has thenucleotide sequence of SEQ ID NO:19.

The invention also provides a vector containing a nucleic acid of theinvention. A vector can further include an expression control sequenceoperably linked to the nucleic acid. The invention additionally provideshost cells comprising such vectors. The invention further provides acomposition that includes such vectors and a pharmaceutically acceptablecarrier.

In yet another aspect, the invention provides a method of eliciting animmune response in an animal. Such a method includes introducing acomposition of the invention into the animal. Such compositions can beadministered orally, intranasally, intraperitoneally, intramuscularly,subcutaneously, or intravenously. Generally, the animal is a swine.

In still yet another aspect, the invention provides a method ofdetermining whether or not an animal has an antibody reactive to animmunogenic polypeptide of the invention, the method comprising:providing a test sample from the animal; contacting the test sample withthe immunogenic polypeptide under conditions permissible for specificbinding of the immunogenic polypeptide with the antibody; and detectingthe presence or absence of the specific binding. Typically, the presenceof specific binding indicates that the animal has the antibody, and theabsence of specific binding indicates that the animal does not have theantibody.

Generally, an appropriate test sample is a biological fluid such asblood, nasal fluid, throat fluid, or lung fluid. In some embodiments,the immunogenic polypeptide is attached to a solid support such as amicrotiter plate, or polystyrene beads. In some embodiments, theimmunogenic polypeptide is labeled. By way of example, the detectingstep can be by radioimmunoassay (RIA), enzyme immunoassay (EIA), orenzyme-linked immunosorbent assay (ELISA).

In another aspect, the invention provides a diagnostic kit for detectingthe presence of an antibody in a test sample, wherein such an antibodyis reactive to an immunogenic polypeptide of the invention. Such a kitcan include one or more of the immunogenic polypeptides of theinvention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is the nucleic acid sequence encoding C2-mhp210 (SEQ ID NO:1), aP102 paralog from M. hyopneumoniae strain 232.

FIG. 2 is the polypeptide sequence of C2-MHP210 (SEQ ID NO:2) from M.hyopneumoniae strain 232.

FIG. 3 is the nucleic acid sequence encoding C2-mhp211 (SEQ ID NO:3)from M. hyopneumoniae strain 232.

FIG. 4 is the polypeptide sequence of C2-MHP211 (SEQ ID NO:4) from M.hyopneumoniae strain 232.

FIG. 5 is the nucleic acid sequence encoding C27-mhp348 (SEQ ID NO:5), aP102 paralog from M. hyopneumoniae strain 232.

FIG. 6 is the polypeptide sequence of C27-MHP348 (SEQ ID NO:6) from M.hyopneumoniae strain 232.

FIG. 7 is the nucleic acid sequence encoding C28-mhp545 (SEQ ID NO:7)from M. hyopneumoniae strain 232.

FIG. 8 is the polypeptide sequence of C28-MHP545 (SEQ ID NO:8) from M.hyopneumoniae strain 232.

FIG. 9 is the nucleic acid sequence encoding C28-mhp662 (SEQ ID NO:9)from M. hyopneumoniae strain 232.

FIG. 10 is the polypeptide sequence of C28-MHP662 (SEQ ID NO:10) from M.hyopneumoniae strain 232.

FIG. 11 is the nucleic acid sequence encoding C28-mhp663 (SEQ ID NO:11),a P102 paralog from M. hyopneumoniae strain 232.

FIG. 12 is the polypeptide sequence of C28-MHP663 (SEQ ID NO:12) from M.hyopneumoniae strain 232.

FIG. 13 is the nucleic acid sequence encoding C2-mhp036 (SEQ ID NO: 13),a P102 paralog from M. hyopneumoniae strain 232.

FIG. 14 is the polypeptide sequence of C2-MPH036 (SEQ ID NO:14) from M.hyopneumoniae strain 232.

FIG. 15 is the nucleic acid sequence encoding C2-mhp033 (SEQ ID NO: 15),a partial paralog of P102 from M. hyopneumoniae strain 232.

FIG. 16 is the polypeptide sequence of C2-MHP033 (SEQ ID NO:16) from M.hyopneumoniae strain 232.

FIG. 17 is the nucleic acid sequence encoding C2-mhp034 (SEQ ID NO: 17),a partial paralog of P102 from M. hyopneumoniae strain 232.

FIG. 18 is the polypeptide sequence of C2-MHP034 (SEQ ID NO:18) from M.hyopneumoniae strain 232.

FIG. 19 is the nucleic acid sequence encoding C28-mhp545 (SEQ ID NO:19)from M. hyopneumoniae strain J.

FIG. 20 is the polypeptide sequence of C28-MHP545 (SEQ ID NO:20) from M.hyopneumoniae strain J.

FIG. 21 is the structure of P102 paralogs and their organization in thechromosome.

FIG. 22 shows a map and hydrophilicity plot of P216. The upper paneldepicts a schematic diagram of the P216 protein sequence. Asterisksindicate locations of peptides used to clone the gene (left, amino acids94-105) and used to make antisera specific for P130 (right, amino acids1654-1668). The arrow indicates the position of the major cleavageevent. The gray box indicates the position of the 30-kDa fragment clonedand expressed (amino acids 1043-1226). The inverted filled triangles arelocations of tryptophan residues encoded by TGA codons. The hatchedboxes are the location of the coiled coil domains. The white boxindicates the location of the BNBD (amino acids 1012-1029). The blackbox represents the transmembrane domain (amino acids 7-30). The lowerpanel represents the hydrophilicity plot.

DETAILED DESCRIPTION

The following abbreviations are used in this application: aa, aminoacid(s); Ab, antibody(ies); bp, base pair(s); CHEF, clamped homogenouselectric field; H., Haemophilus; kb, kilobase(s) or 1000 bp; Kn,kanamycin; LB, Luria-Bertoni media; M., Mycoplasma; mAb, monoclonal Ab;ORF, open reading frame; PCR, polymerase chain reaction; ^(R),resistant/resistance; Tn, transposon(s); ::, novel junction (fusion orinsertion). One letter and three letter code designations for aminoacids are given in Table 1.

TABLE 1 Amino Acid Code Designations Three One letter Letter Amino Acidcode code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic AcidAsp D Cysteine Cys C Glutamic Acid Glu E Glutamine Gln Q Glycine Gly GHistidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K MethionineMet M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val VM. hyopneumoniae Polypeptides and Nucleic Acids

As used herein, the term “polypeptide” refers to a polymer of three ormore amino acids covalently linked by amide bonds. A polypeptide may ormay not be post-translationally modified. As used herein, the term“purified polypeptide” refers to a polypeptide preparation that issubstantially free of cellular material or other contaminatingpolypeptides from the cell or tissue source from which the polypeptideis derived, or substantially free of chemical precursors or otherchemicals when chemically synthesized. For example, a polypeptidepreparation is substantially free of cellular material when thepolypeptide is separated from components of the cell from which thepolypeptide is obtained or recombinantly produced. Thus, a polypeptidepreparation that is substantially free of cellular material includes,for example, a preparation having less than about 30%, 20%, 10%, or 5%(dry weight) of heterologous polypeptides (also referred to herein as a“contaminating polypeptides”). When a polypeptide is recombinantlyproduced, the polypeptide is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,10%, 5% of the volume of the polypeptide preparation. When a polypeptideis produced by chemical synthesis, it is preferably substantially freeof chemical precursors or other chemicals, i.e., it is separated fromchemical precursors or other chemicals that are involved in thesynthesis of the polypeptide. Accordingly, such polypeptide preparationshave less than about 30%, 20%, 10%, 5% (by dry weight) of chemicalprecursors or compounds other than the polypeptide of interest.

As used herein, the term “mutant” refers to a polypeptide, or a nucleicacid encoding a polypeptide, that has one or more conservative aminoacid variations or other minor modifications such that (1) thecorresponding polypeptide has substantially equivalent function whencompared to the wild type polypeptide or (2) an antibody raised againstthe polypeptide is immunoreactive with the wild-type polypeptide.

The term “conservative variation” denotes the replacement of an aminoacid residue by another biologically similar residue, or the replacementof a nucleotide in a nucleic acid sequence such that the encoded aminoacid residue does not change or is another biologically similar residue.Examples of conservative variations include the substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another hydrophobic residue, or the substitution of one polarresidue for another polar residue, such as the substitution of argininefor lysine, glutamic for aspartic acid, or glutamine for asparagine, andthe like. The term “conservative variation” also includes the use of asubstituted amino acid in place of an unsubstituted parent amino acidprovided that antibodies raised to the substituted polypeptide alsoimmunoreact with the unsubstituted polypeptide.

Any M. hyopneumoniae strain may be used as a starting material toproduce the polypeptides and nucleic acids of the present invention.Suitable strains of M. hyopneumoniae may be obtained from a variety ofsources, including depositories such as the American Type CultureCollection (ATCC) (Manassas, Va.) and the NRRL Culture Collection(Agricultural Research Service, U.S. Department of Agriculture, Peoria,Ill.). M. hyopneumoniae strains may also be obtained from lungsecretions or tissues from sick animals followed by inoculating suitableculture media.

An immunogenic polypeptide of the present invention can have an aminoacid sequence shown in FIG. 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.Alternatively, an immunogenic polypeptide of the present invention canbe a fragment of a polypeptide that has an amino acid sequence shown inFIG. 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. An immunogenic polypeptideof the invention can be six or more, or preferably eight or more, aminoacids in length, but less than the full-length number of amino acids.For example, an immunogenic polypeptide can be 10, 12, 15, 20, 25, 30,or greater than 30 amino acids in length. A polypeptide of the presentinvention also can be a mutant of a polypeptide having an amino acidsequence shown in FIG. 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. Mutationsat either the amino acid or nucleic acid level may be useful inimproving the yield of the polypeptides, their immunogenicity orantigenicity, or their compatibility with various expression systems,adjuvants and modes of administration. Synthetic or recombinantfragments of wild type or mutated polypeptides are characterized by oneor more of the antigenic sites of native M. hyopneumoniae polypeptides,the sequences of which are illustrated in FIGS. 2, 4, 6, 8, 10, 12, 14,16, 18, and 20.

The polypeptides of the present invention may be obtained from M.hyopneumoniae cells or may be produced in host cells transformed bynucleic acids that encode these polypeptides. Recombinant polypeptidesproduced from transformed host cells may include residues that are notrelated to M. hyopneumoniae. For example, a recombinant polypeptide maybe a fusion polypeptide containing an amino acid portion derived from anexpression vector, or other source, in addition to the portion derivedfrom M. hyopneumoniae. A recombinant polypeptide may also include astarting methionine. Recombinant polypeptides of the invention displaythe antigenicity of native M. hyopneumoniae polypeptides the sequencesof which are illustrated in FIGS. 2, 4, 6, 8, 10, 12, 14, 16, 18, and20.

Nucleic acid sequences encoding full-length polypeptides of the presentinvention are shown in FIGS. 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19. Thepresent invention encompasses nucleic acid sequences, as well asfragments or mutants of these, that encode immunogenic polypeptides,i.e., capable of eliciting antibodies or other immune responses (e.g.,T-cell responses of the immune system) that recognize epitopes of thepolypeptides having sequences illustrated in FIGS. 2, 4, 6, 8, 10, 12,14, 16, 18, and 20. Hence, nucleic acid sequences of the presentinvention may encode polypeptides that are full-length polypeptides,polypeptide fragments, and mutant or fusion polypeptides.

The term “nucleic acid” as used herein encompasses RNA and DNA,including cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. The nucleic acid can be double-stranded orsingle-stranded. Where single-stranded, the nucleic acid can be thesense strand or the antisense strand. In addition, nucleic acid can becircular or linear.

The term “isolated” as used herein with reference to nucleic acid refersto a naturally-occurring nucleic acid that is not immediately contiguouswith both of the sequences with which it is immediately contiguous (oneon the 5′ end and one on the 3′ end) in the naturally-occurring genomeof the organism from which it is derived. For example, an isolatednucleic acid can be, without limitation, a recombinant DNA molecule ofany length, provided one of the nucleic acid sequences normally foundimmediately flanking that recombinant DNA molecule in anaturally-occurring genome is removed or absent. Thus, an isolatednucleic acid includes, without limitation, a recombinant DNA that existsas a separate molecule (e.g., a cDNA or a genomic DNA fragment producedby PCR or restriction endonuclease treatment) independent of othersequences as well as recombinant DNA that is incorporated into a vector,an autonomously replicating plasmid, a virus (e.g., a retrovirus,adenovirus, or herpes virus), or into the genomic DNA of a prokaryote oreukaryote. In addition, an isolated nucleic acid can include arecombinant DNA molecule that is part of a hybrid or fusion nucleic acidsequence.

The term “isolated” as used herein with reference to nucleic acid alsoincludes any non-naturally-occurring nucleic acid sincenon-naturally-occurring nucleic acid sequences are not found in natureand do not have immediately contiguous sequences in a naturallyoccurring genome. For example, non-naturally-occurring nucleic acid suchas an engineered nucleic acid is considered to be isolated nucleic acid.Engineered nucleic acid can be made using common molecular cloning orchemical nucleic acid synthesis techniques. Isolatednon-naturally-occurring nucleic acid can be independent of othersequences, or incorporated into a vector, an autonomously replicatingplasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), orthe genomic DNA of a prokaryote or eukaryote. In addition, anon-naturally-occurring nucleic acid can include a nucleic acid moleculethat is part of a hybrid or fusion nucleic acid sequence.

It will be apparent to those of skill in the art that a nucleic acidexisting among hundreds to millions of other nucleic acid moleculeswithin, for example, cDNA or genomic libraries, or gel slices containinga genomic DNA restriction digest is not to be considered an isolatednucleic acid.

The term “exogenous” as used herein with reference to nucleic acid and aparticular cell refers to any nucleic acid that does not originate fromthat particular cell as found in nature. Thus, non-naturally-occurringnucleic acid is considered to be exogenous to a cell once introducedinto the cell. It is important to note that non-naturally-occurringnucleic acid can contain nucleic acid sequences or fragments of nucleicacid sequences that are found in nature provided the nucleic acid as awhole does not exist in nature. For example, a nucleic acid moleculecontaining a genomic DNA sequence within an expression vector isnon-naturally-occurring nucleic acid, and thus is exogenous to a cellonce introduced into the cell, since that nucleic acid molecule as awhole (genomic DNA plus vector DNA) does not exist in nature. Thus, anyvector, autonomously replicating plasmid, or virus (e.g., retrovirus,adenovirus, or herpes virus) that as a whole does not exist in nature isconsidered to be non-naturally-occurring nucleic acid. It follows thatgenomic DNA fragments produced by PCR or restriction endonucleasetreatment as well as cDNAs are considered to be non-naturally-occurringnucleic acid since they exist as separate molecules not found in nature.It also follows that any nucleic acid containing a promoter sequence andpolypeptide-encoding sequence (e.g., cDNA or genomic DNA) in anarrangement not found in nature is non-naturally-occurring nucleic acid.

Nucleic acid that is naturally occurring can be exogenous to aparticular cell. For example, an entire chromosome isolated from a cellof person X is an exogenous nucleic acid with respect to a cell ofperson Y once that chromosome is introduced into Y's cell.

Recombinant nucleic acid molecules that are useful in preparing theaforementioned polypeptides are also provided. Preferred recombinantnucleic acid molecules include, without limitation, (1) those havingnucleic acid sequences illustrated in FIGS. 1, 3, 5, 7, 9, 11, 13, 15,17, and 19; (2) cloning or expression vectors containing sequencesencoding recombinant polypeptides of the present invention; (3) nucleicacid sequences that hybridize to those sequences that encode M.hyopneumoniae polypeptides of the invention; (4) degenerate nucleic acidsequences that encode polypeptides of the invention.

Nucleic acids of the invention may be inserted into any of a widevariety of expression vectors by a variety of procedures, generallythrough use of an appropriate restriction endonuclease site. Suitablevectors include, for example, vectors consisting of segments ofchromosomal, non-chromosomal and synthetic nucleic acid sequences, suchas various known derivatives of SV40; known bacterial plasmids, e.g.,plasmids from E. coli including col E1, pCR1, pBR322, pMB9 and theirderivatives; wider host range plasmids, e.g., RP4; phage DNAs, e.g., thenumerous derivatives of phage λ, e.g., NM 989, and other DNA phages suchas M13 or filamentous single stranded DNA phages; yeast plasmids such asthe 2μ plasmid or derivatives thereof; viral DNA such as baculovirus,vaccinia, adenovirus, fowl pox virus, or pseudorabies; and vectorsderived from combinations of plasmids and phage DNAs, such as plasmidswhich have been modified to employ phage DNA or other expression controlsequences.

Within each specific cloning or expression vector, various sites may beselected for insertion of the nucleic acids of this invention. Thesesites are usually designated by the restriction endonuclease that cutsthem, and there are various known methods for inserting nucleic acidsinto these sites to form recombinant molecules. These methods include,for example, dG-dC or dA-dT tailing, direct ligation, synthetic linkers,exonuclease and polymerase-linked repair reactions followed by ligation,or extension of the nucleic acid strand with DNA polymerase and anappropriate single-stranded template followed by ligation. It is to beunderstood that a cloning or expression vector useful in this inventionneed not have a restriction endonuclease site for insertion of thechosen nucleic acid fragment, and that insertion may occur byalternative means.

For expression of the nucleic acids of this invention, these nucleicacid sequences are operatively linked to one or more expression controlsequences in the expression vector. Such operative linking, which may beeffected before or after the chosen nucleic acid is inserted into acloning vehicle, enables the expression control sequences to control andpromote the expression of the inserted nucleic acid.

Any of a wide variety of expression control sequences—sequences thatcontrol the expression of a nucleic acid when operatively linked toit—may be used in these vectors to express the nucleic acid sequences ofthis invention. Such useful expression control sequences include, forexample, the early and late promoters of SV40, the lac or trp systems,the TAC or TRC system, the major operator and promoter regions of λ, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase or other glycolytic enzymes, the promoters of acid phosphatase,e.g., Pho5, the promoters of the yeast α-mating factors, and othersequences known to control the expression of genes in prokaryotic oreukaryotic cells or their viruses, and various combinations thereof. Theexpression vector also includes a non-coding sequence for aribosome-binding site for translation initiation and a transcriptionterminator. The vector may also include appropriate sequences foramplifying expression. In mammalian cells, it is additionally possibleto amplify the expression units by linking the gene to that coding fordehydrofolate reductase and applying a selection to host Chinese hamsterovary cells.

The vector or expression vehicle, and in particular, the sites chosentherein for insertion of the selected nucleic acid fragment, and theexpression control sequence employed in this invention are determined bya variety of factors, e.g., number of sites susceptible to a particularrestriction enzyme, size of the polypeptide to be expressed, expressioncharacteristics such as the location of start and stop codons relativeto the vector sequences, and other factors recognized by those of skillin the art. The choice of a vector, expression control sequence, and/orinsertion site are determined by a balance of these factors, as not allselections are equally effective for a given case.

The recombinant nucleic acid molecule containing the desired codingsequence operatively linked to an expression control sequence may thenbe employed to transform a wide variety of appropriate hosts so as topermit such hosts (transformants) to express the coding sequence, orfragment thereof, and to produce the polypeptide, or portion thereof,for which the hybrid nucleic acid encodes. The recombinant nucleic acidmolecule may also be employed to transform a host so as to permit thathost on replication to produced additional recombinant nucleic acidmolecules as a source of M. hyopneumoniae coding sequences and fragmentsthereof.

A wide variety of hosts are also useful in producing polypeptides andnucleic acids of this invention. These hosts include, for example,bacteria such as E. coli, Bacillus and Streptomyces, fungi such asyeasts, and animal or plant cells in tissue culture. The selection of anappropriate host for these uses is controlled by a number of factors.These include, for example, compatibility with the chosen vector,toxicity of the co-products, ease of recovery of the desiredpolypeptide, expression characteristics, biosafety and costs. Noabsolute choice of host may be made for a particular recombinant nucleicacid molecule or polypeptide from any of these factors alone. Instead, abalance of these factors is applied with the realization that not allhosts may be equally effective for expression of a particularrecombinant nucleic acid molecule.

It is also understood that the nucleic acid sequences that are insertedat the selected site of a cloning or expression vector may includenucleotides that are not part of the actual coding sequence for thedesired polypeptide or may include only a fragment of the entire codingsequence for that polypeptide. It is only required that whatever DNAsequence is employed, the transformed host produces a polypeptide havingthe antigenicity of native M. hyopneumoniae polypeptides.

For example, in an expression vector of this invention, a nucleic acidof this invention may be fused in the same reading frame to a portion ofa nucleic acid sequence coding for at least one eukaryotic orprokaryotic carrier polypeptide or a nucleic acid sequence coding for atleast one eukaryotic or prokaryotic signal sequence, or combinationsthereof. Such constructions may aid in expression of the desired nucleicacid sequence or improve purification, permit secretion, and preferablymaturation of the desired polypeptide from the host cell. The nucleicacid sequence may alternatively include an ATG start codon, alone, ortogether with other codons, fused directly to the sequence encoding thefirst amino acid of a desired polypeptide. Such constructions enable theproduction of, for example, a methionyl or other peptidyl polypeptidethat is part of this invention. This N-terminal methionine or peptidemay then be cleaved intracellularly or extracellularly by a variety ofknown processes or the polypeptide used together with the methionine orother fusion attached to it in the compositions and methods of thisinvention.

The appropriate nucleic acid sequence present in the vector whenintroduced into a host may express part or only a portion of thepolypeptide that is encoded, it being sufficient that the expressedpolypeptide be capable of eliciting an antibody or other immune responsethat recognizes an epitope of the amino acid sequence depicted in FIG.2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. For example, in employing E. colias a host organism, the UGA codon is a stop codon so that the expressedpolypeptide may only be a fragment of the polypeptide encoded by thevector, and therefore, it is generally preferred that all of the UGAcodons in the appropriate nucleic acid sequence be converted intonon-stop codons. Alternatively, an additional nucleic acid sequence thatencodes a t-RNA that translates the UGA codon into a tryptophan residuecan be introduced into the host.

The polypeptide expressed by the host transformed by the vector may beharvested by methods known to those skilled in the art, and used forprotection of a non-human animal such as swine, cattle, etc. againstenzootic pneumonia caused by M. hyopneumoniae. The polypeptide is usedin an amount effective to provide protection against enzootic pneumoniacaused by M. hyopneumoniae and may be used in combination with asuitable physiologically acceptable carrier as described below.

Detecting M. hyopneumoniae

The polypeptides of the present invention may also be used as antigensfor diagnostic purposes to determine whether a biological test samplecontains M. hyopneumoniae antigens or antibodies to these antigens. Suchassays for M. hyopneumoniae infection in an animal typically involveincubating an antibody-containing biological sample from an animalsuspected of having such a condition in the presence of a detectablylabeled polypeptide of the present invention, and detecting binding. Theimmunogenic polypeptide is generally present in an amount that issufficient to produce a detectable level of binding with antibodypresent in the antibody-containing sample.

Thus, in this aspect of the invention, the polypeptide may be attachedto a solid phase support, e.g., a microtiter plate, which is capable ofimmobilizing cells, cell particles or soluble polypeptides. The supportmay then be washed with suitable buffers followed by treatment with thesample from the animal. The solid phase support may then be washed withthe buffer a second time to remove unbound antibody. Labeled polypeptideis added and the support is washed a third time to remove unboundlabeled polypeptide. The amount of bound label on said solid support maythen be detected by conventional means.

By “solid phase support” is intended any support capable of bindingantigen or antibodies. Well-known supports, or carriers, include glass,polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,natural and modified celluloses (especially nitrocellulose),polyacrylamides, agarose, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thepresent invention. The support material may have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to an antigen or antibody. Thus, the support configuration maybe spherical, as in a bead, or cylindrical, as in the inside surface ofa test tube, or the external surface of a rod. Alternatively, thesurface may be flat such as for example, a sheet or test strip.Preferred supports include polystyrene beads.

M. hyopneumoniae specific antibody can be detectably labeled by linkingthe same to an enzyme and using it in an enzyme immunoassay (EIA), orenzyme-linked immunosorbent assay (ELISA). This enzyme, in turn, whenlater exposed to its substrate, will react with the substrate in such amanner as to produce a chemical moiety that can be detected, forexample, by spectrophotometric, fluorometric or by visual means. Enzymesthat can be used to detectably label the M. hyopneumoniae specificantibody include, but are not limited to, horseradish peroxidase, malatedehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeastalcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoa/nylase andacetylcholinesterase.

Detection may be accomplished using any of a variety of immunoassays.For example, by radioactively labeling the recombinant protein, it ispossible to detect antibody binding through a radioimmunoassay (RIA).The radioactive isotope can be detected by such means as the use of agamma counter or a scintillation counter or by autoradiography. Isotopeswhich are particularly useful for the purpose of the present inventioninclude ³H, ¹²⁵I, ¹³¹I, ³⁵S, and ¹⁴C, preferably ¹²⁵I.

It is also possible to label the recombinant polypeptide with afluorescent compound. When the fluorescently labeled polypeptide isexposed to light of the proper wavelength, its presence can then bedetected due to fluorescence. Among the most commonly used fluorescentlabeling compounds are fluorescein isothiocyanate, rhodamine,phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine. The polypeptide can also be detectably labeled usingfluorescence emitting metals such as ¹⁵²Eu, or others of the lanthanideseries. These metals can be attached to the protein using such metalchelating groups as diethylenetriaminepentaacetic acid (DTPA) orethylenediamine-tetraacetic acid (EDTA).

The polypeptide also can be detectably labeled by coupling it to achemiluminescent or bioluminescent compound. The presence of thechemiluminescent-tagged polypeptide is then determined by detecting thepresence of luminescence that arises during the course of a chemicalreaction. Bioluminescence is a type of chemiluminescence found inbiological systems in which a catalytic protein increases the efficiencyof the chemiluminescent reaction. Examples of particularly usefulchemiluminescent labeling compounds are luminol, isoluminol, theromaticacridinium ester, imidazole, acridinium salt and oxalate ester.Important bioluminescent compounds for purposes of labeling areluciferin, luciferase and aequorin.

Detection of the label may be accomplished by a scintillation counter,for example, if the detectable label is a radioactive gamma emitter, orby a fluorometer, for example, if the label is a fluorescent material.In the case of an enzyme label, the detection can be accomplished bycolorimetric methods that employ a substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

The detection of foci of detectably labeled antibodies is indicative ofa disease or dysfunctional state and may be used to measure M.hyopneumoniae in a sample. The absence of such antibodies or otherimmune response indicates that the animal has been neither vaccinatednor infected. For the purposes of the present invention, the bacteriumthat is detected by this assay may be present in a biological sample.Any sample containing it can be used, however, one of the benefits ofthe present diagnostic invention is that invasive tissue removal may beavoided. Therefore, preferably, the sample is a biological fluid suchas, for example, blood, or nasal, throat or lung fluid, but theinvention is not limited to assays using these samples.

In situ detection may be accomplished by removing a histologicalspecimen from an animal, and providing the combination of labeledantibodies of the present invention to such a specimen. The antibody (orfragment) is preferably provided by applying or by overlaying thelabeled antibody (or fragment) to a biological sample. Through the useof such a procedure, it is possible to determine not only the presenceof M. hyopneumoniae but also the distribution of it in the examinedtissue. Using the present invention, those of ordinary skill willreadily perceive that any of a wide variety of histological methods(such as staining procedures) can be modified in order to achieve suchin situ detection.

Alternatively, a sample (e.g., a fluid or tissue sample) may be testedfor the presence of a coding sequence for a M. hyopneumoniae polypeptideof the invention by reaction with a recombinant or synthetic nucleicacid sequence contained within the sequence shown in FIGS. 1, 3, 5, 7,9, 11, 13, 15, 17, 19, or any RNA sequence equivalent to this nucleicacid sequence. The absence of the coding sequence indicates that theanimal has been neither vaccinated nor infected. This test involvesmethods of synthesis, amplification, or hybridization of nucleic acidsequences that are known to those skilled in the art. See, for example,Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2^(nd)Ed, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; PCR, APractical Approach, Vols 1 & 2, McPherson et al. (eds.), OxfordUniversity Press, 1992 and 1995; and PCR Strategies, Innis (ed.),Academic Press, 1995, herein incorporated by reference.

Compositions

The present invention also contemplates a composition (e.g., a vaccine)comprising the recombinant polypeptides of the present invention, ornucleic acid sequences encoding these polypeptides, for immunizing orprotecting non-human animals, preferably swine, against M. hyopneumoniaeinfections, particularly enzootic pneumonia. The terms “protecting” or“protection” when used with respect to the composition for enzooticpneumonia described herein means that the composition prevents enzooticpneumonia caused by M. hyopneumoniae and/or reduces the severity of thedisease. When a composition elicits an immunological response in ananimal, the animal is considered seropositive, i.e., the animal producesa detectable amount of antibodies against a polypeptide of theinvention. Methods for detecting an immunological response in an animalare well known.

Compositions generally include an immunologically effective dosage of apolypeptide of the invention. An “immunologically effective” dosage isan amount that, when administered to an animal, elicits an immunologicalresponse in the animal but does not cause the animal to develop severeclinical signs of an infection. An animal that has received animmunologically effective dosage is an inoculated animal or an animalcontaining an inoculant of an immunologically effective amount of apolypeptide of the invention. Immunologically effective dosages can bedetermined experimentally and may vary according to the type, size, age,and health of the animal vaccinated. The vaccination may include asingle inoculation or multiple inoculations. Other dosage schedules andamounts, including vaccine booster dosages, may be useful.

The composition can be employed in conjunction with a carrier, which maybe any of a wide variety of carriers. Representative carriers includesterile water, saline, buffered solutions, mineral oil, alum, andsynthetic polymers. Additional agents to improve suspendability anddispersion in solution may also be used. The selection of a suitablecarrier is dependent upon the manner in which the composition is to beadministered. The composition is generally employed in non-human animalsthat are susceptible to enzootic pneumonia, in particular, swine.

The composition may be administered by any suitable method, such asintramuscular, subcutaneous, intraperitoneal or intravenous injection.Alternatively, the composition may be administered intranasally ororally, such as by mixing the active components with feed or water, orproviding a tablet form. Methods such as particle bombardment,microinjection, electroporation, calcium phosphate transfection,liposomal transfection, and viral transfection are particularly suitablefor administering a nucleic acid. Nucleic acid compositions and methodsof their administration are known in the art, and are described in U.S.Pat. Nos. 5,836,905; 5,703,055; 5,589,466; and 5,580,859, which areherein incorporated by reference. Other means for administering thecomposition will be apparent to those skilled in the art from theteachings herein; accordingly, the scope of the invention is not limitedto a particular delivery form.

The composition may also include active components or adjuvants (e.g.,Freund's incomplete adjuvant) in addition to the antigen(s) or fragmentshereinabove described. Adjuvants may be used to enhance theimmunogenicity of an antigen. Among the adjuvants that may be used areoil and water emulsions, complete Freund's adjuvant, incomplete Freund'sadjuvant, Corynebacterium parvum, Hemophilus, Mycobacterium butyricum,aluminum hydroxide, dextran sulfate, iron oxide, sodium alginate,Bacto-Adjuvant, certain synthetic polymers such as poly amino acids andco-polymers of amino acids, saponin, iota carrageenan, Regressin™,Avridine™, Mannite monooleate, paraffin oil, and muramyl dipeptide.

Nucleic acid or polypeptide compositions or vaccines as described hereincan be combined with packaging materials including instructions fortheir use to be sold as articles of manufacture or kits. Components andmethods for producing articles of manufactures are well known. Thearticles of manufacture may combine one or more vaccines (e.g., nucleicacid or polypeptide) as described herein. Instructions describing how avaccine is effective for preventing the incidence of a M. hyopneumoniaeinfection, preventing the occurrence of the clinical signs of a M.hyopneumoniae infection, ameliorating the clinical signs of a M.hyopneumoniae infection, lowering the risk of the clinical signs of a M.hyopneumoniae infection, lowering the occurrence of the clinical signsof a M. hyopneumoniae infection and/or spread of M. hyopneumoniaeinfections in animals may be included in such kits.

Conveniently, vaccines of the invention may be provided in apre-packaged form in quantities sufficient for a protective dose for asingle animal or for a pre-specified number of animals in, for example,sealed ampoules, capsules or cartridges.

Application of the teachings of the present invention to a specificproblem or environment is within the capabilities of one having ordinaryskill in the art. Examples of the products and processes of the presentinvention appear in the following examples.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES A. P102 and Paralogs Thereof Example A.1 Mycoplasma Strains

Mycoplasmas hyopneumoniae strains used included the 232, J, andBeaufort. The source and culture conditions used to grow M.hyopneumoniae are as described in Scarman et al. (1997) Microbiology143:663-673.

Example A.2 Cloning of the Gene Encoding P102

The gene encoding P102 was obtained by polymerase chain reaction (PCR)and cloned into pTrcHis (Invitrogen). The oligonucleotides TH130 andTH131 were used to amplify the region encoding amino acids 33 to 887 ofP102 from pISM1217 as described in Hsu and Minion ((1998) Infect. Immun.66:4762-4766). The PCR product having 5′ BamHI and 3′ PstI restrictionenzyme sites was digested sequentially with BamHI and PstI, gelpurified, and ligated into BamHI/PstI-digested pTrcHis plasmid DNA. Theligation mixture was transformed into CSH50 Escherichia coli, andtransformants were selected for ampicillin resistance (100 μg per mL).The resulting plasmid was sequenced with primer SA1528 to confirm theinsertion and orientation of the insert.

Site directed mutagenesis was performed on the insert sequence to removeTGA codons, which code for tryptophan in Mycoplasmas. Directedmutagenesis was performed using the Stratagene QuikChange Site-DirectedMutagenesis Kit (Stratagene, CA) according to the manufacturer'sinstructions. Five TGA codons in the cloned sequence were changed to TGGusing the following primer pairs:

P102.2f: 5′-GAT AAT TTT AAA AAA TGG TCG GCA AAA ACA GTT TTA (SEQ IDNO:21) ACT GCT GCC-3′; P102.2r: 5′-GGC AGC AGT TAA AAC TGT TTT TGC CGACCA TTT TTT (SEQ ID NO:22) AAA ATT ATC-3′; P102.3f: 5′-GAA AGA GGA AGTAAT TGG TTT TCA CGA CTT GAA AGA (SEQ ID NO:23) GC-3′; P102.3r: 5′-GCTCTT TCA AGT CGT GAA AAC CAA TTA CTT CCT CTT (SEQ ID NO:24) TC-3′;P102.4f: 5′-CTA AAA TTC TAA AAT CCT GGC TTG AAA CAA ATC TTC (SEQ IDNO:25) AAG GC-3′; P102.4r: 5′-GCC TTG AAG ATT TGT TTC AAG CCA GGA TTTTAG AAT (SEQ ID NO:26) TTT AG-3′; P102.5f: 5′-GCC TCT CTG ATT ATT GGTATG GAT CTC CGA ATT C-3′; (SEQ ID NO:27) P102.5r: 5′-GAA TTC GGA GAT CCATAC CAA TAA TCA GAG AGG C-3′; (SEQ ID NO:28) P102.6f: 5′-GGG ACA AGC ATTTGG ACA GCT TTT AAT TTC G-3′; (SEQ ID NO:29) P102.6r: 5′-CGA AAT TAA AAGCTG TCC AAA TGC TTG TCC C-3′. (SEQ ID NO:30)

E. coli XL1-Blue MRF′ was the recipient for each mutagenesis step. Toconfirm the sequence and the single-base changes, and to determinewhether errors were introduced during the cloning and mutagenesis steps,the final product was sequenced using the primers:

P102.2-SEQ: 5′-TCC GAC GAT GAC GAT AAG-3′; (SEQ ID NO:31) P102.5-SEQ:5′-TGG AAA ATT AGT TCT TGG-3′; (SEQ ID NO:32) P102.6-SEQ: 5′-AGT TTC CACTTC ATC GCC-3′. (SEQ ID NO:33)

The final construct was designated pISM1316.6.

Example A.3 Expression and Purification of P102

Plasmid pISM1316.6 was transformed into E. coli ER1458 (F-Δ(lac)U169lon-100 hsdR araD139 rpsL(StrR) supF mcrA trp+zjj202::Tn10(TetR)hsdR2(rk-mk+) mcrB1), a Lon protease mutant, in preparation for proteinexpression. An overnight culture was diluted 1:10 into fresh superbrothmedium (per liter; 32 g Bacto tryptone, 20 g yeast extract, 5 g sodiumchloride, pH 7.3) containing 1 mM isopropyl thiogalactopyranoside (IPTG)and protease inhibitor cocktail (Sigma P8848) at a 1:200 dilution. Theculture was incubated for 5 hours at 30° C. with shaking. The cells werecollected by centrifugation and resuspended in TS buffer (10 mM Tris,100 mM sodium chloride, pH 7.4) plus 8 M urea and 2 mg/mL of lysozyme.After incubating for 30 minutes on ice, the suspension was frozen in adry ice ethanol bath and passed sequentially through three freeze-thawcycles. The chromosomal DNA was sheared by passing the suspensionthrough an 18-gauge needle, and insoluble cellular debris was removed bycentrifugation. The final solution was passed through a Talon MetalAffinity Resin (Clontech Laboratories, Inc., CA) column. The column waswashed with 10 column volumes of TS buffer containing 10 mM imidazole.The bound protein was eluted with TS buffer containing 500 mM imidazole,and the column eluent was dialyzed overnight against phosphate bufferedsaline (10 mM Na₂HPO₄, 100 mM NaCl, pH 7.4). Purity of the proteinpreparations was assessed by sodium dodecyl sulfate gel electrophoresisand by Western blotting using 6×His monoclonal antibody (Clontech).

Example A.4 Generation of P102 Antisera

Mice were immunized with 10 μg of purified P102 mixed with 200 μL ofFreund's incomplete adjuvant, and on day 21, second dosages were given.Ascites were developed by the introduction of Sp2 myeloma cells usingthe method of Luo and Lin ((1997) BioTechniques 23:630-632), and ascitesfluid was aliquoted and stored at −70° C. Antibody specificity wastested by immunoblot analysis using purified P102 protein and M.hyopneumoniae whole antigen.

Example A.5 Immunoelectron Microscopic Analysis of Immunogold-labeledCell Sections

To determine if P102 is surface exposed or associated with the P97cilium adhesin, monospecific polyclonal anti-P102 antiserum was used inthe following immunoelectron microscopic studies to determine thelocation of P102 in the Mycoplasma cell.

M. hyopneumoniae strains 90-1 and 60-3 were grown in modified Friismedia (Friis (1971) Acta Vet. Scand. 12:69-79) until mid log phase asdescribed (Hsu et al. (1997) J. Bacteriol. 179:1317-1323). The cellswere pelleted by centrifugation and washed once with phosphate bufferedsaline (PBS) by centrifugation. Cells were resuspended in PBS and thenreacted with either anti-P102 ascite fluid diluted 1:50, or F1B6 cellculture supernatant (Zhang et al. (1995) Infect. Immun. 63:1013-1019)diluted 1:10, overnight at 4° C. The next day, cells were washed fivetimes with PBS and then reacted for 30 minutes at room temperature withgoat anti-mouse IgG+IgM labeled with 10 nm gold particles (EYLaboratories, Inc., San Mateo, Calif.) diluted 1:25. The cells were thenwashed five times with PBS and pelleted by centrifugation.

The final cell pellets were fixed with 3% glutaraldehyde in 0.1 M sodiumcacodylate buffer (pH 7.2) at 4° C. overnight. The pellets were washedthree times, 15 minutes each time, with 0.1 M sodium cacodylate bufferand post fixed with 1% osmium tetroxide in 0.1 M sodium cacodylatebuffer for 2 hours at room temperature. The pellets were then washedwith distilled water, passed through an acetone series and embedded inEmbed 812 and Araldite (Electron Microscopy Sciences, Fort Washington,Pa.).

For tracheal sections, Mycoplasma-free pigs were inoculatedintratracheally with M. hyopneumoniae strain 232 as described in Thackeret al. ((1997) Potentiation of PRRSV pneumonia by dual infection withMycoplasma hyopneumoniae. In Conference of Research Workers in AnimalDiseases. Ellis, R. P. (ed.) Chicago, Ill.: Iowa State University Press,pp. 190). At 10 and 21 days, pigs were sacrificed, and tracheas wereremoved. One cm blocks of tissue were fixed with 1% glutaraldehydeovernight, dehydrated in an acetone series and embedded as above. Thick(1-2 μm) sections were stained with methylene blue polychrome andexamined by microscopy for regions containing ciliated epithelium. Thinsections (80-90 nm) were then prepared for labeling. For some studies,cells grown in vitro were embedded and sectioned prior to staining. Thesections were pretreated with ammonium chloride (1%) for 1 hour, 0.05 Mglycine in PBS for 15 minutes, and blocked for 30 minutes in 2% fishgelatin+2% bovine serum albumin in TS buffer (10 mM Tris, 100 mM NaCl,pH 7.5). Primary antibodies were diluted (1:50) in TS buffer and reactedwith sections for 30 minutes at room temperature. The sections werewashed six times with TS buffer, and then incubated with goat anti-mouseIgG+IgM labeled with 10 nm gold particles (diluted 1:2) for 15 minutesat room temperature. Both primary antibodies and the conjugate werediluted and centrifuged briefly (12,000×g for 5 minutes) to remove goldaggregates prior to use. The sections were then washed six times with TSbuffer, dried, contrasted with osmium vapors for 2 minutes, and stainedwith uranyl acetate-lead citrate. The sections were examined on aHitachi 500 electron microscope at 75 kV.

In in vitro grown cells, gold particles were found external to the cellsand were primarily associated with the extracellular matrix. Similarresults were observed for cells that were stained before or afterfixation and sectioning. Occasionally, particles were seen associatedwith the cell surface, and in rare cases, particles were seenintracellularly. In cells associated with swine cilia, however, goldparticles were seen at high concentration intracellularly. P102 was alsofound in association with swine cilia, often in aggregates or at highconcentrations. The extracellular matrix that was so prominent in brothgrown cells was not evident in sections of infected swine epithelia.

Example A.6 Two-dimensional Electrophoresis

Two-dimensional gel electrophoresis (2-DGE) was carried out essentiallyas described by Guerreiro et al. ((1997) Mol. Plant Microbe Interact.,10:506-16). First dimension immobilized pH gradient (IPG) strips (180mm, linear and non-linear pH 3-10 and linear pH 4-7 and 6-11; AmershamPharmacia Biotech, Uppsala, Sweden) were prepared for focusing bysubmersion in hydration buffer (8 M urea, 0.5% wt/vol CHAPS, 0.2% wt/volDTT, 0.52% wt/vol Bio-Lyte and a trace of bromophenol blue) overnight.M. hyopneumoniae whole cell protein (100 μg for analytical gels, 0.5-1.0mg for preparative gels and immunoblots) was diluted with sample buffer(8 M urea, 4% w/v CHAPS, 1% w/v DTT, 0.8% w/v Bio-Lyte 3-10, 35 mM Tris,and 0.02% w/v bromophenol blue) to a volume of 50 to 100 μL forapplication to the anodic end of each IPG strip. Isoelectric focusingwas performed with a Multiphor II electrophoresis unit (Pharmacia) for200 kVh at 20° C. except for pH 6-11 strips, which were electrophoresedfor 85 kVh. IEF strips were reduced and alkylated in Tris-HCl (0.5 M, pH6.8) containing 6 M urea, 30% w/v glycerol, 2% w/v sodium dodecylsulfate (SDS), 2% w/v DTT and 0.02% bromophenol blue. Equilibratedstrips were placed onto Pharmacia ExcelGels (T=12 to 14% acrylamide) forSDS-PAGE using the Multiphor II. Electrophoretic conditions consisted of200 Volts for 1.5 hours followed by 4 hours at 600 Volts at 5° C. Gelswere stained in Coomassie Blue R-250 (Bio-Rad, Hercules, Calif.), andproteins were transferred to polyvinylidene difluoride (PVDF) membranesusing a Hoefer TE70 Series SemiPhor Semi-Dry Transfer Unit (AmershamPharmacia Biotech, Uppsala, Sweden). The transfer was carried out for1.5 hours at maximum voltage and a current measured by multiplying thearea of the gel (cm²) by 0.8 mA.

Example A.7 Post-separation Analyses

Protein spots were excised from gels using a sterile scalpel and placedin a 96 well tray. Gel pieces were washed with 50 mM ammoniumbicarbonate/100% acetonitrile (60:40 v/v) and then dried in a Speed Vac(Savant Instruments, Holbrook, N.Y.) for 25 minutes. Gel pieces werethen hydrated in 12 μL of 12 ng μL⁻¹ sequencing grade modified trypsin(Promega, Madison, Wis.) for 1 hour at 4° C. Excess trypsin solution wasremoved and the gel pieces immersed in 50 mM ammonium bicarbonate andincubated overnight at 37° C. Eluted peptides were concentrated anddesalted using C₁₈ Zip-Tips™ (Millipore Corp., Bedford, Mass.). Thepeptides were washed on column with 10 μL of 5% formic acid. The boundpeptides were eluted from the Zip-Tip™ in matrix solution (10 mg mL⁻¹α-cyano-4-hydroxycinnamic acid [Sigma] in 70% acetonitrile) directlyonto the target plate. Matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOF MS) mass spectra wereacquired using either a PerSeptive Biosytems Voyager DE-STR (Framingham,Mass.) or a Micromass TofSpec2E (Micromass, Manchester UK). Bothinstruments were equipped with 337 nm nitrogen lasers. All spectra wereobtained in reflectron/delayed extraction mode, averaging 256 lasershots per sample. Two-point internal calibration of spectra wasperformed based upon internal porcine trypsin autolysis peptides (842.5and 2211.10 [M+H]⁺ ions). A list of monoisotopic peaks corresponding tothe mass of generated tryptic peptides was used to search a modifiedtranslated version of the M. hyopneumoniae genome. Successfulidentifications were based on the number of matching peptide masses andthe percentage sequence coverage afforded by those matches. N-terminalEdman sequencing was performed as previously described (Nouwens et al.,2000).

Example A.8 P102 is Surface Expressed

To generate a P102 specific antibody, recombinant P102 protein wasexpressed in in E. coli and then purified as follows. The codingsequence for P102 was obtained from plasmid pISM1217, which containedthe entire sequence of P102 (Hsu and Minion (1998) Infect. Immun.66:4762-4766). The region of the coding sequence encoding amino acids33-887 was amplified by PCR using primers having BamHI and PstIrestriction sites at the 5′ termini to enable cloning into pTrcHis. Theresulting construct was designated pISM1249. To allow for expression ofthe coding sequence in E. coli, the TGA codons in the pISM1249 sequencewere altered by site-directed mutagenesis to TGG codons. The finalconstruct pISM1316.6 was sequenced to confirm these changes and to checkfor errors introduced by PCR during the mutagenesis step.

Expression of the cloned sequence in pISM1316.6 resulted in apoly-histidine-tagged protein of about 100 kDa. Expression levels ofP102 were low in E. coli despite the removal of the opal (TGA) stopcodons. A Talon Metal Affinity Resin column was used to removecontaminating E. coli proteins during purification. Mouse hyperimmuneantiserum raised against this recombinant protein was used in immunoblotanalysis of M. hyopneumoniae whole cells. The anti-P102 antiserum showedthree bands indicating either the presence of cross-reactive proteins orthat P102 was being proteolytically processed. Trypsin treatment ofwhole cells followed by immunoblot and development with the anti-P102antiserum showed that P102 was located on the membrane surface; allimmunoreactive bands were sensitive to trypsin.

Example A.9 P102 Paralogs are Found Throughout the M. hyopneumoniaeGenome

Hybridization studies indicated that P102 or P102-related sequences mayexist in multiple copies in the genome of M. hyopneumoniae (Hsu et al.(1997) J. Bacteriol. 179:1317-1323). Genome sequencing studies haveidentified four distinct paralogs of P102 (C2-mhp210, C27-mhp348,C28-mhp663, and C2-mhp036) and two partial paralogs (C2-mhp033 andC2-mhp034) scattered throughout the chromosome (FIG. 21). Furtheranalysis of the genome sequence of M. hyopneumoniae revealed additionalopen reading frames with varying homologies to P102. Each of theseappeared to be a fusion with a second gene, while the original P102sequence had undergone significant evolution. Also, each paralog waspart of a two-gene genetic structure, possibly organized into operons.In every case, the P102 paralog was the second or downstream gene. DNAsequence analysis of each of the P102 paralogs showed that homology toP102 was low, but amino acid homology was much higher. The amino acidsequences of the P102 paralogs are shown in FIGS. 2, 6, 12, 14, 16,18,and 20.

Example A.10 Biotin Labeling of Surface Accessible Proteins IdentifiedMolecules Belonging to a Multi-gene Family

Studies were undertaken to identify all of the surface accessibleproteins in M. hyopneumoniae recognized by convalescent and hyperimmuneswine sera. By combining surface biotinylation, two-dimensionalimmunoblotting, genomic and proteomic analysis, a subset of thesesurface molecules was mapped to the genome sequence of M. hyopneumoniae.

Initially, two-dimensional gel electrophoresis of biotinylated proteinsidentified groups of proteins that were surface exposed, highlyexpressed, and appeared to resolve along the pI gradient as a series ofspots. The molecular masses of many of these proteins ranged from 40 to130 kDa. Many of these proteins were recognized by convalescent andhyperimmune swine sera. This suggests that these proteins were expressedduring M. hyopneumoniae infection and evoked an accompanying immuneresponse.

Tryptic fragments of individual protein spots were analyzed by peptidemass fingerprinting, and the spectra matched to theoretical trypsincleavage products generated from the M. hyopneumoniae genome database.Some of the spots of different molecular masses mapped to the samesingle copy gene.

Example A.11 Peptide Mass Fingerprinting and Biotinylation Studies Showthat P102 Paralogs are Expressed

Many of the proteins identified by biotinylation and peptide massfingerprinting were related to products from the cilium adhesion operon(Hsu and Minion (1998) Infect. Immun. 66:4762-4766). In addition to thecilium adhesin P97, gene products representing P102 and related proteinswere identified.

A. 12 Results

Results indicated that there were a surprising number of P102 paralogsthat were all expressed and located on the surface of the organism. Someof the P102 paralogs had a greater degree of sequence identity with P97,while other P102 paralogs did not. None of the sequences surrounding theP102 paralogs were similar, which suggests that the P102 genesduplicated and moved independently of surrounding sequences.Differential staining of in vitro-grown and in vivo-grown organisms wasobserved, further suggesting that P102 might be involved in thehyperimmune-like responses seen during infection.

B. P216 Studies Example B.1 Mycoplasma Strains and Culture

The source and culture conditions used to grow M. hyopneumoniae strainsJ, Beaufort and 232 are as described in Scarman et al. ((1997)Microbiology 143:663-673). Mycoplasmas were harvested by centrifugationat 10,000×g, washed three times with TS buffer (10 mM Tris, 150 mM NaCl,pH 7.5), and the final cell pellets were frozen at −20° C. until use.

Example B.2 Preparative Electrophoresis

Preliminary vaccine trials in swine immunised with size-fractionatedantigens of M. hyopneumoniae indicated that antigen pools residing intwo fractions, fractions 2 (85-150 kDa) and 3 (70-85 kDa), providedlimited protection against a virulent challenge (Djordjevic et. al(1997) Aust Vet J 75:504-511). To determine the amino acid sequences ofproteins residing in these molecular mass fractions, whole cell lysatesof M. hyopneumoniae J strain were separated using 5-7% polyacrylamideresolving columns each with a 4% stacking gel using a BioRad 491 PrepCell as described in Scarman et al. ((1997) Microbiology 143:663-673).Proteins corresponding to those defined for fractions 2 and 3 werepooled, concentrated by filtration, and resuspended in PBS. Proteinfractions were digested with trypsin, separated using electrophoresis onprecast 8-15% gradient Tricine gels (Novex), and then blotted onto PVDFmembrane (BioRad, California, USA) (Towbin et al. (1979) Proc. Natl.Acad. Sci. USA. 76:4350-4354). Protein fractions were analyzed by (1)reaction with porcine hyperimmune sera raised against the J strain of M.hyopneumoniae and (2) staining with amido black. Tryptic fragmentsstained with amido black that reacted with the hyperimmune sera wereanalysed by N-terminal amino acid sequencing.

Example B.3 Cloning of the Gene Encoding P216

To clone the genes encoding immunoreactive proteins, degenerateoligonucleotide probes were designed from the N-terminal peptidesequences determined above and used to probe EcoRI-digested chromosomalDNA by Southern analysis (Southern (1975) J. Mol. Biol. 98:503-517).EcoRI digested chromosomal DNA from the Beaufort strain was separated ona 1% agarose column prepared in 491 Prep Cell according to the BioRadTechnical Note #2203. Samples from every fifth fraction were blotted toa nylon membrane and probed with degenerate oligonucleotide probesderived from the N-terminal sequences of tryptic fragments. DNAfragments from reactive fractions were incubated with the Klenowfragment and Pfu DNA polymerase to generate blunt ends. DNA fragmentswere ligated into pCR Script™ and transformed into XL10-Gold as outlinedin the manufacturer's instructions (Stratagene).

In this way, N-terminal sequence analysis of an X kDa tryptic peptidefragment recognised by porcine hyperimmune generated the sequenceELEDNTKLIAPNIRQ (SEQ ID NO:34). Based on this amino acid sequence, adegenerate oligonucleotide having the sequence 5′-GAA (T/C)T(T/A) GAAGAT AAT AC(C/A/T) AAA TTA ATT GC(T/A) CCT AAT-3′ (SEQ ID NO:35) was madeand used as a probe to identify a hybridizing fragment of 4.5 kb. Theclone containing this 4.5 kilobase fragment was designated p216.

Example B.4 DNA Sequence Analysis

For sequence analysis, purified plasmid DNA (Qiagen) or PCR productpurified from agarose using the BRESA-CLEAN™ kit (Bresatec, Adelaide,Australia) was used. Oligonucleotide primers were obtained commercially(Sigma), and the BigDye™ Terminator Cycle Sequencing Kit (AppliedBiosystems) was used for sequencing reactions. Results were analysedwith an Applied Biosystems Model 377 automated sequencer.

Sequence analysis of the cloned fragment in p216 from the Beaufortstrain revealed a large ORF that did not significantly match sequencesdeposited in GenBank. The fragment was the carboxy terminus of a largerORF as the fragment had a stop codon but no ATG start codon. Additionalupstream sequence was obtained by inverse PCR, and the final N-terminalsequence was obtained by PCR using primers designed from strain 232genomic sequences. The complete ORF (C28-mph545; see, FIG. 7) was 5,637base pairs in length and encoded a protein of 216 kDa designated P216(C28-MPH545; see, FIG. 8). The ORF contained 17 TGA codons, 12 of whichappeared in the carboxy terminal 85 kDa.

Blastp analysis of the complete gene sequence revealed near identitywith the partial gene sequence YX2 (GenBank Accession No. AF279292) fromM. hyopneumoniae strain 232 and limited sequence homology with the P97cilium adhesin (GenBank Accession No. U50901) with 21% identities, 38%positives and 19% gaps (Expect=4e-18). Comparisons of the nucleotide andderived protein sequences with the database were performed using thepackage from the University of Wisconsin Genetics Group (GCG) Version 7,accessed via the Australian National Genomic Information Service (ANGIS,University of Sydney) and MacVector (Scientific Imaging Systems, EastmanKodak Co., New Haven, Conn.).

DNA sequence encoding the P216 homologue from the 232 strain of M.hyopneumoniae was obtained as part of a genome-sequencing project.Southern blotting analysis using an oligonucleotide probe from thecarboxy terminus showed that the M. hyopneumoniae genome contained asingle copy of the gene encoding the 216-kDa protein. Blastn analysiswith p216 and the M. hyopneumoniae genome database also identified asingle copy. The protein has 1,879 amino acids, a pI of 8.51, and ishighly hydrophilic. A protein motif search using the algorithm Prositeon the ISREC Profilescan server(www.isrec.isb-sib.ch/software/PFSCAN_form.html) identified a bipartitenuclear binding domain (BNBD) between amino acids 1012-1029.

The nucleotide sequence of the M. hyopneumoniae p216 gene from strain232 and the J strain are shown in FIGS. 7 and 19, respectively.

Example B.5 Generation of Antisera Against M. hyopneumoniae Strain 232

Preparation of porcine hyperimmune serum against M. hyopneumoniae is asdescribed in Scarman et al. (1997) Microbiology 143:663-673. In brief,M. hyopneumoniae-free swines were challenged with a preparation of M.hyopneumoniae strain 232 emulsified in Freund's complete adjuvant, andthese swines were subjected to a second exposure one month later withthe same preparation in Freund's incomplete adjuvant. Serum responseswere monitored until an anti-M. hyopneumoniae response was confirmed byan enzyme-linked immunosorbent assay (ELISA).

Example B.6 Generation of P216 Polyclonal Antisera

To generate monospecific polyclonal antisera to P216, the DNA sequenceencoding P216 from strain 232 was examined for the presence of TGAcodons, since TGA codons encode tryptophans in Mycoplasmas. A regioncontaining no TGA codons and encoding a 30 kDa protein (amino acids1043-1226) was identified. PCR primers were designed to amplify andclone this region into pCR Script™ forming plasmid p216.1. The clonedfragment was then directionally cloned into pQE9 (Qiagen) by ligation ofBamHI- and HindIII-digested p216.1 DNA to form p216.2. The ligationmixture was transformed into Escherichia coli M15[pREP4] according tothe manufacturer's instructions (Qiagen). Colony hybridization using theDIG system (Roche) was used to identify transformants containing theproper fragment.

Cultures of the transformants containing p216.2 were grown in LB medium(Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2^(nd)Ed, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) containingampicillin (100 μg/mL) and kanamycin (25 μg/mL) at 37° C. with shaking.For expression from p216.2, cultures were treated with 1 mMisopropyl-β-D-thiogalactopyranoside (IPTG) after reaching an OD₆₀₀ of0.6. After induction for 4 hours, the cells were harvested bycentrifugation at 4,000×g for 20 minutes. Purification of therecombinant His-tagged protein was achieved using Ni-NTA resin underdenaturing conditions as outlined in the manufacturer's instructions(Qiagen).

Purified recombinant protein was dialysed against PBS containing 5%glycerol and concentrated using polyvinyl-pyrrolidone (Sigma).Approximately 5 mg of purified protein in a volume of 250 μL wereemulsified with an equal volume of Freund's incomplete adjuvant (Sigma).The preparation was given subcutaneously to rabbits at two sites and abooster immunization, similarly prepared, was given three weeks later.Serum response against the immunizing antigen was confirmed byimmunoblot analysis.

Similarly, rabbit antisera directed against the N-terminal sequence ofP216 were generated by immunization with the peptide DFLTNNGRTVLE (SEQID NO:36) (amino acids 94-105 of P216) conjugated to keyhole limpethemocyanin. Rabbit immunizations were performed as described in (Scarmanet al. (1997) Microbiology 143:663-673).

Example B.7 Electrophoretic and Immunoblot Analyses

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)and immunoblot analysis were performed as described by Laemmli (1970)Nature 227:680-685 and Towbin et al. (1979) Proc. Natl. Acad. Sci. USA,76:4350-4354, respectively. Analytical electrophoretic gels containingM. hyopneumoniae strain 232 proteins were stained with silver (Rabilloudet al. (1992) Electrophoresis 13:264-266). Preparative gels were stainedwith colloidal Coomassie Brilliant Blue G-250 (0.1% Coomassie BrilliantBlue G-250 w/v, 17% w/v ammonium sulfate, 34% methanol v/v, 3% v/vortho-phosphoric acid). Gels were destained in 1% v/v acetic acid for 1hour.

Immunoblot analysis was used to determine if P216 is recognised byantibodies elicited during natural infection using swine field serashown to contain antibodies against M. hyopneumoniae (Djordjevic et al.(1994) Vet. Microbiol. 39:261-273). The 30 kDa recombinant proteinrepresenting amino acids 1043-1226 of P216 was used as antigen in theseexperiments. Other immunoblot analyses included one- and two-dimensionalblots of M. hyopneumoniae whole cells using swine convalescent serapools (2D blots) and individual swine sera (1D blots). Swine hyperimmunesera were also used to screen for immunoreactive proteins in one- andtwo-dimensional immunoblot analyses. Rabbit antisera generated againstthe 30 kDa recombinant protein and the peptide DFLTNNGRTVLE (SEQ IDNO:36) specific for P130 were used to investigate processing of P216 inone-dimensional immunoblotting experiments as well.

Example B.8 Two-dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis was carried out essentially asdescribed by Guerreiro et al. ((1997) Mol Plant Microbe Interact10:506-516). First dimension immobilized pH gradient (IPG) strips (180mm, linear and non-linear pH 3-10 and linear pH 4-7;Pharmacia-Biotechnology, Uppsala, Sweden) were prepared for focusing bysubmersion in rehydration buffer (8 M urea, 0.5% w/v CHAPS, 0.2% w/vDTT, 0.52% w/v Bio-Lyte and a trace of bromophenol) overnight. M.hyopneumoniae 232 whole cell proteins (100 μg for analytical gels,0.5-1.0 mg for preparative gels and immunoblots) were diluted withsample buffer (8 M urea, 4% w/v CHAPS, 1% w/v DTT, 0.8% w/v Bio-Lyte3-10, 35 mM Tris, and 0.02% w/v bromophenol blue) to a volume of 50 to100 μl for application to the anodic end of each IPG strip. Isoelectricfocusing was run with the Immobiline DryStrip kit in a Multiphor IIelectrophoresis unit (Pharmacia-Biotechnology) for 200 kVh at 20° C. IEFstrips were subsequently prepared for second dimensionSDS-polyacrylamide gel electrophoresis (SDS-PAGE) by equilibration inTris-HCl (0.5 M, pH 6.8) containing 6 M urea, 30% w/v glycerol, 2% w/vsodium dodecyl sulfate (SDS), 2% w/v DTT, and 0.02% bromophenol blue.Equilibrated strips were placed onto Pharmacia ExcelGel gels (T=12 to14% acrylamide) for molecular mass separation of M. hyopneumoniaeproteins on a Multiphor II unit. Electrophoretic conditions consisted of200 Volts for 1.5 hour followed by 4 hours at 600 Volts. Gels weremaintained at 5° C. throughout.

Example B.9 Peptide Mass Fingerprinting-mass Spectrometry

Proteins spots were manually excised and placed in a 96-well microtiterplate. Conditions used for trypsin digestion and for the generation ofpeptide mass fingerprints are described in Nouwens et al. (2000)Electrophoresis 21:3797-3809. A purification step was performed on thetryptic peptides for proteins with poor peptide mass fingerprints asdescribed in Gobom et al. (1999) J. Mass Spectrom. 34:105-116. Proteinidentifications were assigned by comparing the peak lists generated frompeptide mass fingerprinting data to a database containing theoreticaltryptic digests of M. hyopneumoniae strain 232. The Protein-Lynx package(Micromass, Manchester, UK) was used to search databases.

Example B.10 Image Processing

Gels and immunoblots were digitized at 600 dpi with a UMAX PS-2400X lampscanner using Photoshop 3.0 (Adobe, Mountain View, Calif.). Spotdetection and gel-to-gel protein spot matching were performed withMELANIE II software (BioRad, Hercules, Calif.) run under OpenWindows3.0. Apparent molecular masses were determined by co-electrophoresiswith protein standards (Pharmacia-Biotechnology).

Example B.11 Results of Two-dimensional Electrophoresis and Peptide MassFingerprinting Analysis

Analyses of two-dimensional electropherograms identified two clusters ofspots that tracked along the pI gradient in an unusual fashion. Peptidemass fingerprinting analysis of spots within each of the clusters showedthat the spots had identical mass fingerprints and were thus derivedfrom the same molecule. Cluster 1 with an approximate mass of 130 kDawas mapped to the N-terminal region of P216 from the genome sequence ofM. hyopneumoniae strain 232. Cluster 2 of approximately 85 kDa mapped tothe carboxy terminus of the same ORF. The proteins were designated P130and P85, respectively. The pI of cluster 1 ranged from 9.5 to 8.0, whilethe p1 of cluster 2 ranged from 9.0 to 6.5. Mass spectrometric analysisindicated that P216 was cleaved between amino acids 1004 and 1090generating the two fragments of 130 and 85 kDa.

Example B.12 Results of Immunoblot Analysis

Two-dimensional immunoblots reacted with porcine hyperimmune serarevealed a complex pattern of spots two of which corresponded to P130and P85. P85 was also strongly recognized by a pool of convalescent serashowing that it was an important antigen during disease. To investigatethis further, a 30-kDa region spanning amino acids 1042-1226 in P85 wasexpressed, purified by nickel-affinity chromatography, and blotted ontoPVDF membrane. Individual convalescent sera from swines known to bepositive in a M. hyopneumoniae-specific ELISA reacted with the 30-kDaprotein confirming that P216 is an important molecule recognized by thehost immune response during the normal course of infection. Antibodiesraised to a 30-kDa peptide spanning amino acids 1042-1226 reacted solelywith the 85 kDa cleavage product suggesting that cleavage occurredbetween amino acids 1004 and 1042. Sera raised to the N-terminal peptideof P216 recognized only P130

Example B.13 Posttranslational Processing of P216 Among DifferentStrains of M. hyopneumoniae

To investigate fragment patterns of P216 in different M. hyopneumoniaestrains, immunoblot analysis was performed with the anti-P130 N-terminalpeptide and anti-P30 antisera. Antibodies raised against the N-terminalpeptide recognized P130 and several lower molecular mass peptides inone-dimensional immunoblots of whole cell lysates of J and 232 strains.The pattern of proteins recognised by this antisera was differentbetween the two strains. Antisera raised against the 30-kDa peptidestrongly recognised an 85-kDa antigen in both J and 232 strains, butalso reacted with a number of weakly reactive proteins. Similarly, thepattern recognised with the anti-30-kDa sera was different between J and232.

To determine if different post-translational cleavage events wereoccurring among other strains of M. hyopneumoniae, a collection ofstrains from different geographic origins were examined by immunoblot.Anti-30 kDa sera reacted strongly to an 85-kDa antigen and otherproteins of lower molecular mass in immunoblots of whole cell lysatesfrom different strains of M. hyopneumoniae. These strains representedisolates recovered from different geographic locations within Australiaand from different countries including the USA, Great Britain andFrance. The anti-P30 sera, however, did not react against antigens inimmunoblots of whole cell lysates of related porcine Mycoplasmas, e.g.Mycoplasma hyorhinis and Mycoplasma flocculare, suggesting that P216 isa M. hyopneumoniae-specific antigen. Convalescent sera from differentswines also recognized purified recombinant P30 indicating that P216 isexpressed in vivo.

Example B.14 Surface Localization Studies

Several approaches were taken to determine if P216 and its cleavageproducts were associated with the outer membrane surface. These includedtrypsin digestion and cell surface biotinylation.

For trypsin digestion studies, all solutions and M. hyopneumoniae cellstocks were pre-equilibrated at 37° C. M. hyopneumoniae cells (200 mg/mLin PBS) were aliquoted (300 μL) into sterile eppendorf tubes at 37° C.and trypsin was added to a final concentration ranging from 0.1-1000μg/mL. The suspensions were inverted gently and incubated at 37° C. for20 minutes. Immediately after incubation, the cells were lysed inLaemmli buffer, heated at 95° C. for 10 minutes and analysed by SDS PAGEand immunoblotting. Trypsin digested both P85 and P130 in aconcentration dependent manner, but did not digest the intracellularenzyme lactate dehydrogenase, a control for spontaneous lysis of cells(Strasser et al. (1991) Infect. Immun. 59:1217-22). This suggests thatboth portions of P216 are surface accessible and sensitive to trypsindigestion.

To further clarify this, surface biotinylation of M. hyopneumoniae wasperformed. The method described by Meier et al. ((1992) Anal. Biochem.204:220-226) was used with the following modifications. All solutionswere pre-chilled at 4° C. and all manipulations were performed on ice.M. hyopneumoniae pellets (200 mg wet weight) were resuspended in 4 mL ofBOS buffer (10 mM sodium tetraborate in 0.15 M NaCl, pH 8.8).Immediately after the addition of 5 μL of NHS-biotin (10 mg/mL indimethylsulfoxide), the reaction was allowed to proceed for 1 to 8minutes with swirling. To determine the most suitable reaction time,aliquots were removed at 1-minute intervals for 15 minutes. A reactiontime of 5 minute was chosen for all subsequent studies except wherenoted. Biotinylation was stopped with the addition of 2 mL of 0.1 MNH₄Cl that served to saturate unbound NHS-biotin. Cells were harvestedby centrifugation (8,500×g, 10 minutes) and washed twice in TKMS buffer(25 mM Tris-HCl, pH 7.4, 25 mM KCl, 5 mM MgCl₂ and 0.15 M NaCl in PBS).The products were resolved by two-dimensional electrophoresis.

Both P130 and P85 were readily biotinylated, confirming that all partsof P216 were surface accessible.

Example B.15 Triton X-100 and X-114 Extractions

Integral membrane proteins from 200 mg wet weight of whole cells wereextracted with TX-114 essentially as described by Bordier ((1981) J.Biol. Chem. 182:1356-1363). The resultant aqueous and detergent phaseswere collected and analysed by SDS-PAGE and immunoblotting. The phasepartitioning activity of Triton X-114 causes separation of hydrophobicmolecules into the detergent phase. When treated with Triton X-114, P85remained in the insoluble pellet consisting of complex high molecularweight structures that (1) were membrane associated and (2) lacked thesolubility of normal cytosolic proteins.

For Triton X-100 extraction, pelleted M. hyopneumoniae (strains J andBeaufort) cells (200 mg wet weight) were resuspended in 10 mL of TSbuffer containing 1 mM phenylmethylsulfonyl fluoride. Proteins wereextracted by the addition of 2% Triton X-100 (Amersham PharmaciaBiotechnology) and incubated at 37° C. for 30 minutes as described inStevens and Krause ((1991) J. Bacteriol 173:1041-1050). Briefly, M.hyopneumoniae cell suspensions were centrifuged (14,000×g, 30 min) at 4°C. The aqueous phase was removed and the pellet was re-extracted asdescribed above. The insoluble pellet and both aqueous phases wereanalysed by SDS-PAGE and immunoblotting using anti-30 kDa and seraraised against the peptide DFLTNNGRTVLE (SEQ ID NO:36).

With Triton X-100 fractionation, high molecular weight cytoskeletal-likeproteins remain insoluble, but phase partitioning does not occur. Whentreated with Triton X-100, P85 partitioned primarily to the aqueousdetergent-containing phase, but about 30% remained in the pellet. Thesedata indicate that P216 may form extracellular oligomeric structures.The presence of coiled coil domains in both fragments of P216 alsosupports this hypothesis.

C. P97 Studies Example C.1 Bacterial Strains and Plasmids

M. hyopneumoniae strains 232 (virulent parental strain), 232_(—)91.3(high adherent clone), 232_(—)60.3 (low adherent clone), and J typestrain (NCTC 10110) were grown in modified Friis broth and harvested asdescribed by Zhang et al. ((1995) Infect Immun 63:1013-1019) andDjordjevic et al. ((1994) Vet Microbiol 39:261-273), respectively. Allbroth media were filter sterilized through 0.22 μm filters, whichremoved the majority of particulate matter. Mycoplasmas were harvestedby centrifugation and extensively washed to remove remaining mediumcontaminants. Escherichia coli TOP10 containing pISM405 was grown onLuria Bertani (LB) agar or in LB broth (Sambrook et al., 1989)containing 100 μg ml⁻¹ ampicillin. Isopropyl-β-D-thiogalactopyranoside(IPTG) induction was carried out by the addition of IPTG to a finalconcentration of 1 mM. Bacterial cultures were routinely grown at 37° C.and liquid cultures were aerated by shaking at 200 rpm.

Example C.2 Construction and Expression of Adhesin Fusion Protein

Hexa-histidyl P97 fusion proteins were constructed using the pTrcHis(Invitrogen, Carlsbad, Calif.) cloning vector. Primers FMhp3 (5′-GAA CAATTT GAT CAC AAG ATC CTG AAT ATA CC-3′ (SEQ ID NO:37)) and RMhp4 (5′-AATTCC TCT GAT CAT TAT TTA GAT TTT AAT TCC TG-3′ (SEQ ID NO:38)) were usedto amplify a 3013 bp fragment representing base pairs 315-3321 of thegene sequence containing amino acids 105-1107. The fragment was digestedwith BclI (underlined sequence) and inserted into the BamHI site ofvector pTrcHisA. A construct with the proper fragment orientation wasidentified by restriction digests. The resulting 116-kDa recombinantP97-polyhistidine fusion protein contained the R1 and R2 repeat regionsas well as the major cleavage site at amino acid 195 in the P97sequence.

Example C.3 Antisera

The Mab F1B6 has been described (Zhang et al. (1995) Infect. Immun.63:1013-1019). Mab F1B6 binds to the R1 region of the cilium adhesinthat has at least 3 repeat sequences (Minion et al. (2000) Infect.Immun. 68:3056-3060). Peptides with sequences TSSQKDPST (ΔNP97) (SEQ IDNO:39) and VNQNFKVKFQAL (NP97) (SEQ ID NO:40) were used to raiseantibodies against P97/P66 and P22, respectively. The peptides werebound to keyhole limpet hemocyanin with the Pierce Imjet MaleimideActivated Immunogen Conjugation Kit (Pierce Chemical Co., Rockford,Ill.). These conjugates were then used to generate mouse hyperimmuneantisera by the method of Luo and Lin ((1997) BioTechniques 23:630-632).The resulting antisera were tested by enzyme linked immunosorbent assay(ELISA) using ovalbumin-peptide conjugate and purified recombinant P97antigens, and by immunoblot with the recombinant P97 antigen. Antiserumraised against the C-terminal 28 kDa (R2 serum) of the cilium adhesin ofstrain J has been described (Wilton et al. (1998) Microbiology144:1931-1943). Mouse Mab 2B6-D4 raised against human fibronectin waspurchased commercially (BD Biosciences, Pharmingen) as was alkalinephosphatase conjugated goat anti-mouse Ig(H+L) antibodies (SouthernBiotechnology Associates, Inc., Birmingham, Ala.). Goat anti-mouseIgG+IgM labeled with 10 nm colloidal gold particles (EY Laboratories,Inc., San Mateo, Calif.) was used in immunogold electron microscopystudies.

Example C.4 Immunoblot Analysis

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) andimmunoblot analysis was performed as described by Laemmli ((1970) Nature227:680-685) and Towbin et al. ((1979) Proc. Natl. Acad. Sci. USA.76:4350-4354), respectively. Proteins were transferred to PVDF membranes(Micron Separations, Inc.). For the media control experiments, purifiedrecombinant P97 was incubated with fresh and spent Friis media. Spentmedia was prepared from an early log phase culture that had beencentrifuged and filtered through a 0.1 μm filter. Purified recombinantP97 (2.5 μg) in 20 μl phosphate buffered saline was diluted 1:1 in freshor spent media and incubated overnight at 37° C. Ten μl of the mixturewere the loaded onto SDS-PAGE gels, blotted to nitrocellulose anddeveloped with F1B6 Mab. For ligand blotting, PVDF blots weretransferred, blocked and washed as described previously (Wilton et al.(1998) Microbiology 144:1931-1943). Blots were exposed to humanfibronectin (5 μg ml⁻¹) dissolved in TS buffer (TS buffer: 10 mMTris-HCl, pH 7.4; 150 mM NaCl) for 1.5 h, washed, and exposed to 0.4 μgml⁻¹ anti-human fibronectin Mabs for 1 h at room temperature. Blots werewashed and developed as described above.

Example C.5 Trypsin Treatment of M. hyopneumoniae

M. hyopneumoniae cells (0.5 g) were treated with trypsin essentially asdescribed previously (Wilton et al. (1998) Microbiology 144:1931-1943).Briefly, trypsin was added to cell suspensions of M. hyopneumoniae at 0,0.3, 0.5, 1.0, 3.0, 10, 50, 300, and 500 μg ml⁻¹ at 37° C. for 15 min.Immediately after incubation, cell suspensions were lysed in Laemmlibuffer and heated to 95° C. for 10 min. Lysates were analysed bySDS-PAGE and immunoblotting using F1B6 Mab.

Example C.6 Two-dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis (2-DGE) was carried out essentiallyas described by Cordwell et al. ((1997) Electrophoresis 18:1393-1398).First dimension immobilized pH gradient (IPG) strips (180 mm, linearpH6-11; Amersham Phamracia Biotech, Uppsala, Sweden) were prepared forfocusing by submersion in 2-DGE compatible sample buffer (5 M urea, 2 Mthiourea, 0.1% carrier ampholytes 3-10, 2% w/v CHAPS, 2% w/vsulfobetaine 3-10, 2 mM tributyl phosphine (TBP; Bio-Rad, Hercules USA))overnight. M. hyopneumoniae whole cell protein (250 μg)) was dilutedwith sample buffer to a volume of 100 μl for application to the anodicend of each IPG strip via an applicator cup. Isoelectric focusing wasperformed with a Multiphor II electrophoresis unit (Amersham PharmaciaBiotech) for 85 kVh at 20° C. IPG strips were detergent exchanged,reduced and alkylated in buffer containing 6 M urea, 2% SDS, 20%glycerol, 5 mM TBP, 2.5% v/v acrylamide monomer, trace amount ofbromophenol blue dye and 375 mM Tris-HCl (pH 8.8) for 20 minutes priorto loading the IPG strip onto the top of an 8-18% T, 2.5% C (piperazinediacrylamide) 20 cm×20 cm polyacrylamide gel. Second-dimensionelectrophoresis was carried out at 4° C. using 3 mA/gel for 2 hours,followed by 20 mA/gel until the bromophenol blue dye had run off the endof the gel. Gels were fixed in 40% methanol, 10% acetic acid for 1 hourand then stained overnight in Sypro Ruby (Molecular Probes, Eugene,Oreg.). Images were acquired using a Molecular Imager Fx (Bio-Rad). Gelswere then double-stained in Coomassie Blue G-250.

Example C.7 Post-separation Analyses

Protein spots were excised from gels using a sterile scalpel and placedin a 96 well tray (Gobom et al. (1999) J. Mass. Spectrom. 34:105-116).Gel pieces were washed with 50 mM ammonium bicarbonate/100% acetonitrile(60:40 v/v) and then dried in a Speed Vac (Savant Instruments, Holbrook,N.Y.) for 25 min. Gel pieces were then hydrated in 12 μl of 12 ng μl⁻¹sequencing grade modified trypsin (Promega, Madison, Wis.) for 1 h at 4°C. Excess trypsin solution was removed and the gel pieces immersed in 50mM ammonium bicarbonate and incubated overnight at 37° C. Elutedpeptides were concentrated and desalted using C₁₈ Zip-Tips™ (MilliporeCorp., Bedford, Mass.). The peptides were washed on a column with 10 μl5% formic acid. The bound peptides were eluted from the Zip-Tip™ inmatrix solution (10 mg ml⁻¹ α-cyano-4-hydroxycinnamic acid [Sigma] in70% acetonitrile) directly onto the target plate. Matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)mass spectra were acquired using either a PerSeptive Biosytems VoyagerDE-STR (Framingham, Mass.) or a Micromass TofSpec2E (Micromass,Manchester UK). Both instruments were equipped with 337 nm nitrogenlasers. All spectra were obtained in reflectron/delayed extraction mode,averaging 256 laser shots per sample. Two-point internal calibration ofspectra was performed based upon internal porcine trypsin autolysispeptides (842.5 and 2211.10 [M+H]⁺ ions). A list of monoisotopic peakscorresponding to the mass of generated tryptic peptides was used tosearch a modified translated version of the M. hyopneumoniae genome.Successful identifications were based on the number of matching peptidemasses and the percentage sequence coverage afforded by those matches.N-terminal Edman sequencing was performed as previously described(Nouwens et al. (2000) Electrophoresis 21:3797-3809).

Example C.8 Immunoelectron Microscopy

M. hyopneumoniae strain 232 cells were grown to mid log phase, pelletedby centrifugation and washed with phosphate buffered saline (PBS). Thefinal cell pellets were fixed with 3% glutaraldehyde in 0.1 M sodiumcacodylate buffer (pH 7.2) at 4° C. overnight. The pellets were washedthree times with 0.1 M sodium cacodylate buffer, 15 min between changesand post fixed with 1% osmium tetroxide in 0.1 M sodium cacodylatebuffer for 2 h at room temperature. The pellets were then washed withdistilled water, passed through an acetone series and embedded in Embed812 and Araldite (Electron Microscopy Sciences, Fort Washington, Pa.).Thin sections (80-90 nm) were then washed six times with TS buffer, andreacted with F1B6 ascites fluid (diluted 1:50), anti-ΔNP97 ascites fluid(diluted 1:10), anti-NP97 ascites fluid (diluted 1:10), or mouseanti-human fibronectin (diluted 1:25) overnight at 4° C. The grids werewashed five times with TS buffer and then reacted with goat anti-mouseIgG+IgM labeled with 10 nm colloidal gold particles (EY Laboratories,Inc.) diluted 1:25 for 30 min at room temperature. The cells were thenwashed 5 times with TS buffer, dried, contrasted with osmium vapors for2 min, and stained with uranyl acetate-lead citrate. The sections wereexamined on a Hitachi 500 at 75 kV.

For tracheal sections, mycoplasma-free pigs were inoculatedintratracheally with M. hyopneumoniae strain 232. At 10 and 21 days,pigs were sacrificed, tracheas were removed and 1 cm blocks of tissuefixed with 1% glutaraldehyde overnight, dehydrated in an acetone series,and embedded as above. Thick (1-2 μm) sections were stained withmethylene blue polychrome and examined by microscopy for regionscontaining ciliated epithelium. Thin sections (80-90 nm) were thenprepared for labeling. The sections were pretreated with ammoniumchloride (1%) for 1 h, 0.05 M glycine in PBS for 15 min, blocked for 30min in 2% fish gelatin+2% bovine serum albumin in TS buffer (10 mM Tris,100 mM NaCl, pH 7.5). Primary antibodies were diluted in TS buffer andreacted with sections for 30 min at room temperature. The sections werewashed six times with TS buffer, and then incubated with goat anti-mouseIgG+IgM labeled with 10 nm gold particles (diluted 1:2) for 15 min atroom temperature. Both primary antibodies and the conjugate were dilutedand centrifuged briefly (12,000×g for 5 min) prior to use. The sectionswere then washed six times with TS buffer, dried, contrasted with osmiumvapors for 2 min, and stained with uranyl acetate-lead citrate. Thesections were examined on a Hitachi 500 at 75 kV.

Example C.9 Fibronectin Binding Assay

Immunlon 2 (Dynatech Laboratories, Inc.) 96 well plates were coated with100 μl of human fibronectin (Sigma, F 0895) at a concentration of 5 μgml⁻¹ in 0.1 M sodium carbonate. Plates were incubated at 4° C.overnight, washed three times with PBS, and blocked with 1% bovine serumalbumin in PBS for 2 hr. The plates were then incubated with purifiedrecombinant P97 with or without inhibitor at a concentration of 10 μgml⁻¹. Inhibitors tested were intact human fibronectin, 45-kDaproteolytic fragment of fibronectin (Sigma, F 0162), 30-kDa proteolyticfragment of fibronectin (Sigma, F 9911) and engineered RGD polymer(Sigma, 5022). They were added to Eppendorf tubes with purifiedrecombinant P97 (10 μg ml⁻¹) at concentrations of 37.5 μg ml⁻¹, 7.5 μgml⁻¹, and 1.5 μg ml⁻¹ and incubated at 37° C. for 1 hr. The recombinantP97 plus inhibitor was then transferred to a fibronectin coated plate,which was then incubated at 37° C. for 2 hr. Binding of P97 tofibronectin was assessed by ELISA with Mab F1B6. Optical density at 405nm was indicative of P97 binding to fibronectin-coated wells. Threereplicates per treatment were assayed from three different experiments.Statistical differences were determined by the General Linear Model witha linear contrast based on pooled variances.

Example C.10 Results of Two-dimensional Gel Electrophoresis and MassSpectrometry

Previous studies have demonstrated that the gene product for the ciliumadhesin of strain 232 (126-kDa preprotein, 1036 amino acids) undergoes acleavage event at amino acid 195 to yield what was once thought to bethe “mature” molecule (Hsu et al. (1997) J. Bacteriol. 179:1317-1323).During peptide mass mapping studies of J strain proteins, four spots of22, 28, 66 and 94 kDa (subsequently referred to as P22, P28, P66 andP94, respectively) were identified that represented different fragmentsof the adhesin. The N-terminal sequences for these proteins allowedunequivocal alignment with the cilium adhesin preprotein. P94 of strainJ, the homologue of P97 in strain 232, mapped to a region that beginsimmediately downstream of amino acid 195 until the end of the ORF. Twoclosely spaced proteins at 66 kDa had identical mass maps andcorresponded to a region beginning immediately downstream of amino acid195 of the adhesin and ending near the R1 repeat. N-terminal sequenceanalysis of P66 showed a sequence of ADEKTSS (SEQ ID NO:41) that isidentical to that of P94. Immunoblotting results using Mab F1B6confirmed that P66 contains R1. Thus, the cleavage event must occurimmediately downstream of the R1 repeat region. These data suggest thata fragment approximately 28 kDa in size had been removed from theC-terminus in some, but not all of the P94 molecules. This observationwas confirmed when a 28-kDa fragment was identified that mapped to theC-terminus of P94. Also, one and two-dimensional immunoblots of J strainproteins probed with antisera raised against a recombinant 28-kDaprotein containing R2 but not R1 (Wilton et al. (1998) Microbiology144:1931-1943) recognised both P28 and P94 proteins. Previously, it wasshown that antisera raised against a 28-kDa C-terminal recombinantpeptide of the adhesin recognised the mature form of this antigen (93-97kDa) in different strains of M. hyopneumoniae and a 28-kDa fragment onlyin strain J (Wilton et al. (1998) Microbiology 144:1931-1943). Trypticpeptide mass mapping showed that peptides from P22 mapped to the first190 amino acids of the 123-kDa adhesin preprotein. The N-terminalsequence of P22 (SKKSKTF (SEQ ID NO:42)) aligned to amino acids 2-8 inthe N-terminus of the 123 kDa preprotein suggesting that cleavage of thehydrophobic leader peptide (amino acids 8-22) is not necessary fortranslocation of the cilium adhesin across the membrane.

Comparative peptide mass mapping studies of strain 232 identified twospots of 70 and 97 kDa, subsequently identified as P70 and P97,respectively. Mass maps representative of P97 corresponded to a regionbeginning immediately downstream of amino acid 195 until the end of theORF and corresponded to the most abundant product of the 232 strainadhesin gene (Zhang et al. (1995) Infect. Immun. 63:1013-1019).Interestingly, mass maps representative of P70 corresponded to a regionbeginning immediately downstream of amino acid 195 and ending near theR1 repeat, a map that was virtually identical to P66 in strain J. Thepresence of six extra copies of the R1 repeat is the most likelyexplanation for the difference in masses between P66 and P70 in strainsJ and 232, respectively. Consistent with these data, immunoblots probedwith antisera raised against a recombinant 28-kDa protein containing R2but not R1 (Wilton et al. (1998) Microbiology 144:1931-1943) recognizedP97 but not P70 or P28. Furthermore, P28 or P22 could not be identifiedon 2D gels of 232 proteins resolved by 2D gel electrophoresis in regionswhere they were identified in strain J. This variation was not due todifferences in sequence since P22 sequences were identical in the twostrains. This was not true for the P28 sequences, however. The predictedmass and pI for P28 from strain 232 was 24.6 kDa and 5.88, respectively,and for P28 from strain J, it was 26.0 kDa and 8.39. It was possiblethat P28 was not found in strain 232 because of the change in pI causinga shift in the gel location of the protein. It was also possible thatadditional cleavage of P22 occurred in strain 232 that did not in strainJ.

To rule out the possibility that cleavage resulted from a proteolyticactivity in the media used for culturing M. hyopneumoniae, purifiedrecombinant P97 was incubated with fresh and spent medium and thenexamined for proteolytic cleavage by immunoblot. Because the mediumcontained 20% swine serum, large quantities of swine immunoglobulinswere present in the protein samples causing some background stainingwith the anti-mouse conjugate. It was still clear, however, that neitherfresh nor spent medium contained proteolytic activity capable ofcleaving recombinant P97 after 12 hours of incubation at 37° C. Thus,cleavage of the cilium adhesin was mediated by mycoplasma-encodedactivities and was not due to porcine serum or other medium components.

Example C.11 Trypsin Sensitivity of R1-containing Cleavage Products

Immunoblot analyses of strain J and 232 cells digested with differentconcentrations of trypsin was used to investigate the cellular locationof R1-containing cleavage fragments. The F1B6 Mab typically recognisedproteins with masses of 35, 66, 88, 94, and 123 kDa in strain J and asimilar pattern was observed for strain 232. Exposure of intact M.hyopneumoniae to concentrations of trypsin ranging from 0.1-10 μg ml⁻¹showed a gradual loss of the higher mass proteins. Concentrationsbetween 10 and 50 μg ml⁻¹ resulted in the loss of all the immunoreactiveproteins (except one of 35 kDa) indicating that R1-containing adhesinfragments are surface accessible. The pattern of digestion ofR1-containing adhesin fragments was consistent in repeat experimentsexcept that the 35 kDa fragment was not reliably resistant to trypsin atconcentrations above 10 μg ml⁻¹. Identical blots reacted with antiseraraised to recombinant M. hyopneumoniae lactate dehydrogenase (previouslyshown to reside cytosolically) (Strasser et al. (1991) Infect. Immun.59:1217-1222) and to antisera raised to recombinant fragments ofpyruvate dehydrogenase subunits A and D showed that these proteinsremained detectable with trypsin concentrations up to 500 μg ml⁻¹. Incontrol experiments where lysed cells were exposed to trypsin, lactatedehydrogenase and pyruvate dehydrogenase subunit D were rapidlydegraded.

Example C.12 Results of Immunogold Electron Microscopy

Transmission electron microscopy studies have shown that high and lowadherent strains of M. hyopneumoniae differ in their outer membranestructure. High adherent clones possessed fibrils on the outer surfacethat appeared to interconnect to adjacent cells; these fibrils wererarely observed in low adherence clones (Young et al. (1994) Isolationand characterization of high and low adherent clones of Mycoplasmahyopneumoniae. In IOM Letters. 10^(th) International Congress of theInternational Organization for Mycoplasmology. Vol. 3 Bordeaux, France,pp. 684-685). Antisera generated against specific regions of the adhesinenabled analysis of cleavage in vivo using immunogold electronmicroscopy. Virulent strain 232 was used in these studies because theseresults would have the most impact on understanding pathogenicmechanisms. R1-specific Mab F1B6 and antisera raised to peptidesTSSQKDPST (ΔNP97 antiserum) (SEQ ID NO:39) and VNQNFKVKFQAL (NP97antiserum) (SEQ ID NO:40) were used in these studies. The Mab F1B6remained associated with the mycoplasma membrane, but not intimatelyassociated with the cell confirming a previous report (Zhang et al.(1995) Infect. Immun. 63:1013-1019) and the trypsin studies above. ΔNP97antiserum showed that this portion of the molecule is located distal tothe membrane in association with extracellular material of unknowncomposition. In some instances, the antibodies seemed to definefibrial-like structures still attached to the mycoplasma cell membrane.NP97 antibodies clustered in aggregates to cytosolic locations,intimately to the membrane surface, and were also observed at sitesdistant from the extracellular surface of the cell membrane.

Example C.13 Fibronectin Binding Results

Since cleavage of the cilium adhesin occurs at amino acid position 195(Hsu et al. (1997) J. Bacteriol. 179:1317-1323), it was not readilyapparent how the remaining adhesin could remain associated with the celland direct binding to porcine cilia. Immunogold studies showed that allcilium binding R1 epitopes remained cell associated in the absence ofthe hydrophobic N-terminus sequence, but apparently are not inserteddirectly into the membrane. This is not surprising since no other regionof the protein has sufficient hydrophobicity to direct membraneinsertion (Hsu et al. (1997) J. Bacteriol. 179:1317-1323). Thepossibility that other proteins may play a role in bridgingR1-containing protein fragments of the cilium adhesin to the membranethrough protein-protein interactions was examined. Analysis of thepredicted protein sequence of the 123 kDa adhesin preprotein with thecomputer program COILS (http://www.ch.embnet.org) revealed that theprotein contained three coiled coil domains. One of these residedbetween amino acids 180-195 in P22 (14-, 21- and 28-amino acid windowsettings) and two were located in P97 between amino acids 367-387(window setting 14) and 780-805 (window setting 14 and 21). Thesedomains are known to mediate protein-protein interactions. In addition,it was thought that the R1 and R2 domains might also play a role ininteractions with other proteins. One obvious protein to test wasfibronectin, a protein found in abundance throughout the host and shownto participate in other bacterial-host interactions (Probert et al.(2001) Infect. Immun. 69:4129-4133; Talay et al. (2000) Cell Microbiol.2:521-535; Rocha and Fischetti (1999) Infect. Immun. 67:2720-2728; andSchorey et al. (1996) Mol. Microbiol. 21:321-329).

Ligand blotting studies confirmed that recombinant P97 bound porcinefibronectin. Other fibronectin binding proteins were also identified inlysates of M. hyopneumoniae low (lane 1) and high (lane 2) adherentvariants of strain 232 and in strain J (lane 3). The low and highadherent strains of 232 differed by the absence of a fibronectin-bindingband at approximately 50 kDa, which was also present in strain J.

Fibronectin binding assays with human fibronectin and purifiedrecombinant cilium adhesin were also performed. Maximum inhibitionoccurred with the engineered RGD domain at all three concentrationstested (p<0.001). Inhibition also occurred with intact fibronectin(p<0.001) as expected. Interestingly, the 45-kDa purified fragment offibronectin enhanced binding at the highest concentration tested.

To investigate the role(s) fibronectin might play in the binding of M.hyopneumoniae to porcine respiratory epithelial cells, anti-fibronectinantibodies were applied to lung sections showing M. hyopneumoniae strain232 in close association with respiratory epithelial cilia. Goldparticles were localised in regions where M. hyopneumoniae cells wereintimately associated with cilia, on the surface of cilia and on thesurface of M. hyopneumoniae cells.

D. Detection of Infection and Immunogenic Compositions Example D.1Detection of M. hyopneumoniae Infection in Swine

The polypeptides displaying M. hyopneumoniae antigenicity of thisinvention may be used in methods and kits designed to detect thepresence of M. hyopneumoniae infection in swine herds and therefore torecognize swine in a herd which have been infected by this bacteria. Forexample, the antigens produced by hosts transformed by recombinantnucleic acid molecules of this invention, or antibodies raised againstthem, can be used in RIA or ELISA for these purposes. In one type ofradioimmunoassay, antibody against one or more of the antigens of thisinvention, raised in a laboratory animal (e.g., rabbits), is attached toa solid phase, for example, the inside of a test tube. Antigen is thenadded to the tube to bind with the antibody.

A sample of swine serum, taken from 1 of each 10 to 20 swine per herd,together with a known amount of antigen antibody labeled with aradioactive isotope, such as radioactive iodine, is then added to thetube coated with the antigen-antibody complex. Any antigen (a marker forM. hyopneumoniae infection) antibody in the swine serum will competewith the labeled antibody for the free binding sites on antigen-antibodycomplex. Once the serum has been allowed to interact, the excess liquidis removed, the test tube washed, and the amount of radioactivitymeasured. A positive result, i.e., that the tested swine's serumcontains M. hyopneumoniae antibody, is indicated by a low radioactivecount.

In one type of ELISA test, a microtiter plate is coated with one or moreantigens of this invention and to this is added a sample of swine serum,again, from 1 in every 10 or 20 swine in a herd. After a period ofincubation permitting interaction of any antibody present in the serumwith the antigen, the plate is washed and a preparation of antigenantibodies, raised in a laboratory animal and linked to an enzyme label,is added, incubated to allow reaction to take place, and the plate isthen rewashed. Thereafter, enzyme substrate is added to the microtiterplate and incubated for a period of time to allow the enzyme to work onthe substrate, and adsorbance of the final preparation is measured. Alarge change in adsorbance indicates a positive result, i.e., the testedswine serum had antibodies to M. hyopneumoniae and was infected withthat bacteria.

Example D.2 Immunogenic Compositions

Standard methods known to those skilled in the art may be used inpreparing immunogenic compositions of polypeptides and nucleic acids ofthe present invention for administration to swine. For example, thepolypeptide of choice may be dissolved in sterile saline solution. Forlong-term storage, the polypeptide may be lyophilized and thenreconstituted with sterile saline solution shortly beforeadministration. Prior to lyophilization, preservatives and otherstandard additives such as those to provide bulk, e.g., glycine orsodium chloride, may be added. A compatible adjuvant may also beadministered with the composition.

In addition, compositions can be prepared using antibodies raisedagainst the polypeptides of this invention in laboratory animals, suchas rabbits. This “passive” vaccine can then be administered to swine toprotect them from M. hyopneumoniae infection. Direct incorporation ofnucleic acid sequences into host cells may also be used to introduce thesequences into animal cells for expression of antigen in vivo.

The above description, drawings and examples are only illustrative ofpreferred embodiments that achieve the objects, features and advantagesof the present invention. It is not intended that the present inventionbe limited to the illustrated embodiments. Any modification of thepresent invention that comes within the spirit and scope of thefollowing claims should be considered part of the present invention.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A purified immunogenic polypeptide, the amino acid sequence of whichcomprises SEQ ID NO:
 8. 2. A composition comprising the immunogenicpolypeptide of claim
 1. 3. A diagnostic kit for detecting the presenceof an antibody in a test sample, wherein said antibody is reactive tothe immunogenic polypeptide of claim 1, said kit comprising theimmunogenic polypeptide of claim
 1. 4. A method of eliciting an immuneresponse in an animal, said method comprising introducing thecomposition of claim 2 into said animal.
 5. The method of claim 4,wherein said composition is administered orally, intranasally,intraperitoneally, intramuscularly, subcutaneously, or intravenously. 6.The method of claim 4, wherein said animal is a swine.
 7. A method ofdetermining whether or not an animal has an antibody reactive to theimmunogenic polypeptide of claim 1, said method comprising: providing atest sample from said animal; contacting said test sample with saidimmunogenic polypeptide under conditions permissible for specificbinding of said immunogenic polypeptide with said antibody; anddetecting the presence or absence of said specific binding, wherein saidpresence of specific binding indicates that said animal has saidantibody, and wherein said absence of specific binding indicates thatsaid animal does not have said antibody.
 8. The method of claim 7,wherein said test sample is a biological fluid.
 9. The method of claim8, wherein said biological fluid is selected from the group consistingof blood, nasal fluid, throat fluid, and lung fluid.
 10. The method ofclaim 7, wherein said immunogenic polypeptide is attached to a solidsupport.
 11. The method of claim 10, wherein said solid support is amicrotiter plate, or polystyrene beads.
 12. The method of claim 7,wherein said immunogenic polypeptide is labeled.
 13. The method of claim7, wherein said detecting is by radioimmunoassay (RIA), enzymeimmunoassay (EIA), or enzyme-linked immunosorbent assay (ELISA).